Metallic nanoparticle synthesis with carbohydrate capping agent

ABSTRACT

The disclosure relates to metal nanoparticle compositions and their methods of formation and use, in particular gold nanoparticles (AuNP) and gold-coated magnetic nanoparticles. Compositions according to the disclosure include aqueous suspensions of metal nanoparticles that are stabilized with one or more carbohydrate capping agents and/or that are functionalized with one or more binding pair members for capture/detection of a target analyte. The nanoparticle suspensions are stable for extended periods and can be functionalized as desired at a later point in time, typically prior to use in an assay for the detection of a target biological analyte. The stable nanoparticle suspension can be formed by the aqueous reduction of oxidized metal precursors at non-acidic pH values in the presence of a carbohydrate-based capping agent such as dextrin or other oligosaccharides.

CROSS REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Application Nos. 61/557,644(filed on Nov. 9, 2011) and 61/674,485 (filed Jul. 23, 2012), both ofwhich are incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under 2007-ST-061-000003awarded by the U.S. Department of Homeland Security. The government hascertain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED IN A COMPUTER READABLE FORMAT

The application contains nucleotide sequences which are identified withSEQ ID NOs. The Sequence Listing provided in computer readable form,incorporated herein by reference in its entirety, is identical to thewritten copy of the Sequence Listing provided with the application.

BACKGROUND OF THE DISCLOSURE

Field of the Disclosure

The disclosure generally relates to the formation of metal nanoparticles(e.g., gold nanoparticles) using a carbohydrate capping agent (e.g.,dextrin). The metal nanoparticles can be in the form of a metalnanoparticle core stabilized by the carbohydrate capping agent.Alternatively, the metal nanoparticles can be in the form of ananoparticle core having a metal coating in a core-shell configuration,where the metal shell is stabilized by the carbohydrate capping agent.The metal nanoparticles are formed by reduction of a metal precursor ata neutral or alkaline pH in the presence of the carbohydrate cappingagent, and optionally a nanoparticle core material. The carbohydratecapping agent provides a stabilized aqueous suspension of the metalnanoparticles. The metal nanoparticles can be used for detection of ananalyte by functionalization of the nanoparticles with a binding pairmember specific to the target analyte of interest.

Brief Description of Related Technology

Gold nanoparticles (AuNPs) have attracted considerable interest inrecent years due to their wide range of application, for example insensing methods as tracers and transducers. The spectral, electrical andchemical properties make AuNPs suited for sensing, molecular labeling,and bio-engineering (Li et al. 2002; Rechberger et al. 2003). Sensingtechnologies utilizing AuNPs include oxidation-reduction potentiometry,conductors in electrical circuits, and spectral reporters (2-100 nm) insolution (Dudak 2009; Pal et al. 2007; Xiliang et al. 2006). The sensingmethodologies using AuNPs rely on attached surface biomolecules asrecognition materials. The ligands (DNA, proteins, polymer, peptides,and antibodies) act as capping agents to stabilize the AuNP in aqueoussolution and provide surface functionality, biological capture andchemical reactivities (Chah et al. 2005; Goluch et al. 2006; Hill andMirkin 2006; Slocik et al. 2005; Zhou et al. 2009).

The most common AuNP synthesis techniques utilize citrate under acidicreaction conditions. Common methodologies for AuNP synthesis are: 1)non-polar synthesis using the Brust method (Brust et al. 1994), 2)aqueous generation with the Turkevich method (Turkevich et al. 1951),and 3) biological synthesis using microbial agents (Ahmad et al. 2003;Bharde et al. 2007; Das et al. 2009). Aqueous generation has been understudy due to the “greener” nature of water-based reactions in comparisonto non-polar solvents. The basic chemistry of formation for citratereduction techniques is to reduce the Au³⁺ ion to Au⁰ and stabilize thesurface of the colloidal gold with a capping molecule that is soluble inthe synthesis media (Daniel and Astruc 2004). Traditional aqueoussynthesis involves low pH and/or high temperatures; which limit thenumber of biological capping agents, requiring a ligand exchange stepafter synthesis for sensing applications.

Neutral to alkaline synthesis methods have been explored using microbialsynthesis (Bharde et al. 2007; Das et al. 2009), sodium hydroxidereduction (Zhou et al. 2009) and sodium borohydride reactions (Brust etal. 1994). The appeal of microbial synthesis is that biological ligands(proteins, carbohydrates, glyco-lipids) are present during generation inan aqueous medium and could be used as the capping agent but have notshown the same control, stability and consistency of generation ascurrent techniques for AuNP production (Torres-Chavolla 2010). Severalbiological agents have been explored for the reduction and capping of[AuCl₄]⁻ to produce gold colloids, including cysteine (Ma and Han 2008),tryptophan (Selvakannan et al. 2004), and ascorbic acid (Andreescu etal. 2006). The standard Burst technique of sodium borohydride formationrequires a non-polar generation and a phase exchange for water solublefunctionalization. Recent studies have explored the use of polymers forcapping agents with sodium hydroxide as the reduction agent; but havenot explored biomolecule attachments (Zhou et al. 2009). The reaction pHin most of these methods is within the acidic range and the resultingAuNP size is between 30-80 nm. Limited exploration of citrate has beenconducted in alkaline conditions but reported as extremely slow andstill requires post production ligand exchange (Ji et al. 2007).

Glyconanoparticles (carbohydrate functionalized nanoparticles) haverecently been explored for carbohydrate-carbohydrate andcarbohydrate-protein interaction studies, and for applications inbiomedicine, including bio-labeling and biosensors (Aslan et al. 2005;de la Fuente and Penades 2006). Gold glyconanoparticles (AuGlycoNP) canbe synthesized using a modification of the Brust methodology using oneof several mono and disaccharides (e.g. lactose, maltose, and glucose)(de la Fuente and Penades 2006) for post production attachment with aligand exchange technique. Recent work has successfully usedcyclodextrin, dextrin, and glucose as aqueous capping agents afterorganic media production (Huang et al. 2004; Porta and Rossi 2003).

SUMMARY

The disclosure relates to alkaline metallic nanoparticle generationprocedures, for example in the 7-11 pH range. Longer reaction times(e.g., on the order of hours), can permit the introduction ofbio-molecular capping agents during synthesis. The disclosed process isgenerally an aqueous, alkaline synthetic process for the formation ofmetal nanoparticles (e.g., AuNPs or a (magnetic) nanoparticle core witha gold shell) using a carbohydrate or oligosaccharide (e.g., dextrin)capping agent during generation. The carbohydrate capping agent is aremovable capping agent that is compatible with standard post-productionfunctionalization methodologies for the metal nanoparticles, for exampleusing thiolated ligands to functionalize a gold surface. The reactionrates for nanoparticle formation are dependent on pH, temperature, andcapping agent concentration, which factors contribute to control thereaction speed of the process as well as the particle-size distributioncharacteristics of the formed nanoparticles.

In one aspect, the disclosure relates to a method for method for formingmetal nanoparticles, the method comprising: (a) providing an aqueousmedium, the aqueous medium comprising (i) water and (ii) metal ions insolution in the water; and (b) reducing the metal ions in the aqueousmedium at a neutral or alkaline pH value (e.g., pH 7-12) in the presenceof a carbohydrate capping agent (e.g., dextrin) for a time sufficient toform a plurality of reduced metal nanoparticles as a suspensionstabilized in the aqueous medium with the carbohydrate capping agent.

In another aspect, the disclosure relates to method for forming goldnanoparticles, the method comprising (a) providing an aqueous medium,the aqueous medium comprising (i) water and (ii) gold ions (e.g., insolution or otherwise mixed in the water); and (b) reducing the goldions in the aqueous medium at a pH value ranging from 8 to 11 in thepresence of a dextrin capping agent for a time sufficient to form aplurality of reduced gold nanoparticles as a suspension stabilized inthe aqueous medium with the dextrin capping agent; wherein the pluralityof reduced gold nanoparticles has an average particle size ranging from5 nm to 15 nm. In a refinement, the metal ion reduction in part (b) isperformed in the presence of galactose in addition to the dextrincapping agent.

In another aspect, the disclosure relates to a stabilized metalnanoparticle suspension composition comprising: (a) water in sufficientamount to provide an aqueous medium; and (b) a plurality of stabilizedmetal nanoparticles stably suspended in the aqueous medium, eachstabilized metal nanoparticle comprising: (i) a metal nanoparticle coreand (ii) a carbohydrate capping agent present as a layer on an outersurface of the metal nanoparticle core in an amount sufficient tostabilize the metal nanoparticle suspension. In a refinement, thestabilized metal nanoparticles are capable of remaining stably suspendedin the aqueous medium for a period of at least 90 days when stored atroom temperature. In another refinement, each stabilized metalnanoparticle further comprises a binding pair member (A) immobilized onthe outer surface of the metal nanoparticle core and (B) capable ofbinding to a target analyte.

In another aspect, the disclosure relates to a method forfunctionalizing stabilized metal nanoparticles, the method comprising:(a) providing the stabilized metal nanoparticle suspension according toany of the various disclosed embodiments (e.g., dextrin-stabilized goldor other metal nanoparticles); (b) providing a binding pair membercomprising (i) an immobilization moiety for immobilizing the bindingpair member onto the metal nanoparticle and (ii) a binding moietycapable of binding to a target analyte or a second binding pair member;and (c) performing a ligand exchange process between the stabilizedmetal nanoparticle suspension and the binding pair member, thereby (i)removing at least some of the carbohydrate capping agent present as alayer on the outer surface of the metal nanoparticle and (ii)immobilizing the binding pair member on the outer surface of the metalnanoparticle via the immobilization moiety. In a refinement, the metalnanoparticles comprise gold nanoparticles; and the binding pair membercomprises (A) a ssDNA oligonucleotide as the binding moiety, the ssDNAbeing capable of specific binding to a target ssDNA analyte and (B) athiol functional group as the immobilization moiety. In anotherrefinement, the metal nanoparticles comprise gold nanoparticles; and thebinding pair member comprises (B) a carboxylate functional group as thebinding moiety and (B) a thiol functional group as the immobilizationmoiety.

In another aspect, the disclosure relates to a method for forming metalnanoparticles, the method comprising: (a) providing an aqueous medium,the aqueous medium comprising (i) water, (ii) metal ions in solution inthe water, and (iii) core nanoparticles dispersed in the water; and (b)reducing the metal ions in the aqueous medium at a neutral or alkalinepH value in the presence of a carbohydrate capping agent for a timesufficient to form a plurality of reduced metal nanoparticles as asuspension stabilized in the aqueous medium with the carbohydratecapping agent, the reduced metal nanoparticles comprising a metalcoating from the reduced metal ions on the core nanoparticles. In anextension, the method further comprises: (c) magnetically separating (i)reduced metal nanoparticles comprising the metal coating on the corenanoparticles from (ii) reduced metal nanoparticles formed without acore nanoparticle interior. In an embodiment, the core nanoparticlescomprise magnetic nanoparticles, and the reduced metal nanoparticlescomprise metal-coated magnetic nanoparticles. In another embodiment, themetal ions comprise gold ions, the core nanoparticles comprise ironoxide nanoparticles, and the metal nanoparticles comprise gold-coatediron oxide nanoparticles.

In another aspect, the disclosure relates to a stabilized metalnanoparticle suspension composition comprising: (a) water in sufficientamount to provide an aqueous medium; and (b) a plurality of stabilizedmetal nanoparticles stably suspended in the aqueous medium, eachstabilized metal nanoparticle comprising: (i) a nanoparticle core, (ii)a metal coating on the nanoparticle core, and (iii) a carbohydratecapping agent present as a layer on an outer surface of the metalcoating in an amount sufficient to stabilize the metal nanoparticlesuspension. In an embodiment, the nanoparticle core comprises a magneticmaterial. In another embodiment, the nanoparticle core comprises an ironoxide and the metal coating on the nanoparticle core comprises gold. Ina refinement, each stabilized metal nanoparticle further comprises abinding pair member (A) immobilized on the outer surface of the metalcoating and (B) capable of binding to a target analyte.

In another aspect, the disclosure relates to a method forfunctionalizing stabilized metal nanoparticles, the method comprising:(a) providing a stabilized metal nanoparticle suspension according toany of the various disclosed embodiments (e.g., including a (magnetic)nanoparticle core); (b) providing a binding pair member comprising abinding moiety capable of binding to a target analyte or a secondbinding pair member; and (c) performing a ligand exchange processbetween the stabilized metal nanoparticle suspension and the bindingpair member, thereby (i) removing at least some of the carbohydratecapping agent present as a layer on the outer surface of the metalnanoparticle and (ii) immobilizing the binding pair member on the outersurface of the metal nanoparticle. In an embodiment, the binding pairmember comprises an antibody capable of specific binding to a targetbacterial or viral analyte. In another embodiment, the binding pairmember comprises an immunoconjugate binding pair member, theimmunoconjugate comprising: (i) an immunoglobulin comprising (A) an Fcregion and (B) an antigen-binding region being capable of specificallybinding to the target analyte, and (ii) an immunoglobulin-bindingprotein having a binding affinity to the Fc region of theimmunoglobulin, the immunoglobulin-binding protein being immobilized onthe outer surface of the metal nanoparticle and bound to the Fc regionof the immunoglobulin. In a refinement, (i) the immunoglobulin-bindingprotein can be selected from the group consisting of protein A, proteinG, protein A/G, and combinations thereof, and/or (ii) the immunoglobulincan be selected from the group consisting of IgA, IgD, IgE, IgG, IgM,subclasses thereof, and combinations thereof. In another embodiment, thecarbohydrate capping agent comprises dextrin, the nanoparticle corecomprises an iron oxide, and the metal coating on the nanoparticle corecomprises gold.

In another aspect, the functionalized stabilized metal nanoparticles canbe used in a method for detecting the target analyte, the methodcomprising: (a) forming a conjugate between the target analyte and thebinding pair member of the functionalized stabilized metal nanoparticles(e.g., by contacting the components in a sample matrix), (b) optionallymagnetically separating the analyte conjugate from the sample matrix(e.g., when the metal nanoparticles include a magnetic nanoparticlecore), and (c) detecting the metal component of the nanoparticlescorresponding to the analyte conjugate (e.g., electrochemicallydetecting the gold or other metallic portion of the nanoparticulateanalyte conjugate).

In another aspect, the disclosure relates to a method for formingmagnetic gold nanoparticles, the method comprising: (a) providing anaqueous medium, the aqueous medium comprising (i) water, (ii) gold ionsin solution in the water, and (iii) magnetic iron oxide nanoparticlesdispersed in the water; and (b) reducing the gold ions in the aqueousmedium at a pH value ranging from 8 to 11 in the presence of a dextrincapping agent for a time sufficient to form a plurality of reducedmagnetic gold nanoparticles as a suspension stabilized in the aqueousmedium with the dextrin capping agent, the magnetic gold nanoparticlescomprising a gold coating from the reduced gold ions on the magneticiron oxide nanoparticles; wherein the plurality of reduced magnetic goldnanoparticles has an average particle size ranging from 5 nm to 20 nm.

In another aspect, the disclosure relates to a method for detecting atarget analyte, the method comprising: (a) providing a functionalized,stabilized metal nanoparticle comprising: (i) a metal nanoparticle, (ii)a carbohydrate capping agent (e.g., dextrin) present as a layer on anouter surface of the metal nanoparticle (e.g. in an amount sufficient tostabilize the metal nanoparticle as a suspension in an aqueous medium),and (iii) a binding pair member (A) immobilized on the outer surface ofthe metal nanoparticle and (B) capable of binding to a target analyte;(b) forming an analyte-nanoparticle conjugate between a target analyteand the binding pair member of the functionalized, stabilized metalnanoparticle (e.g., by contacting the two components in a sample matrixcontaining the target analyte); and (c) detecting a metal component(e.g., gold) of the metal nanoparticle of the analyte-nanoparticleconjugate (e.g., in the presence of the carbohydrate capping agent;detection or lack thereof of the metal component can be correlated to acorresponding positive or negative detection of the target analyte inthe original sample matrix). In a refinement, part (c) compriseselectrochemically detecting the metal component of the metalnanoparticle in the presence of the carbohydrate capping agent. Inanother refinement, (i) the analyte-nanoparticle conjugate furthercomprises a magnetic moiety and (ii) the method further comprisesmagnetically separating the analyte-nanoparticle conjugate from a samplematrix in which the analyte-nanoparticle conjugate is formed prior todetecting the metal component thereof. In another refinement, thebinding pair member comprises an antibody capable of specific binding toa target bacterial analyte or a target viral analyte. In anotherrefinement, the binding pair member comprises an immunoconjugate bindingpair member as described above. In an embodiment, (i) the metalnanoparticle comprises a metal nanoparticle core formed from the metalcomponent (e.g., a gold nanoparticle providing gold/gold ions as thecomponent to be detected), and (ii) the carbohydrate capping agent ispresent as a layer on an outer surface of the metal nanoparticle core,In another embodiment, (i) the metal nanoparticle comprises (A) ananoparticle core (e.g., a magnetic material such as an iron oxide) and(B) a metal coating on the nanoparticle core (e.g., a gold coatingproviding gold/gold ions as the component to be detected), the metalcoating being formed from the metal component, and (ii) the carbohydratecapping agent is present as a layer on an outer surface of the metalcoating.

Various refinements and extensions of the foregoing methods andcompositions are possible. For example, the metal ions can comprise goldions and the metal nanoparticles can comprise gold nanoparticles. Moregenerally, the metals for the metal ions and corresponding metalnanoparticles/metal shells can be selected from the group consisting ofgold, chromium, copper, zinc, nickel, cadmium, silver, cobalt, indium,germanium, tin, lead, arsenic, antimony, bismuth, chromium, molybdenum,manganese, iron, ruthenium, rhodium, palladium, osmium, iridium,platinum, alloys thereof, and combinations thereof. The aqueous mediumcan further comprise a counter ion in solution in the water from adissolved metal ionic compound providing the metal ions. Reduction ofthe metal ions can be performed at a temperature ranging from 20° C. to100° C. In a refinement, the aqueous medium further comprises a combinedreducing agent for reducing the metal ions and pH-adjusting agent (e.g.,a carbonate such as sodium carbonate) for maintaining the neutral oralkaline pH value of the aqueous medium during reduction. Thecarbohydrate capping agent can comprise an oligosaccharide having 3 to100 saccharide residues. The carbohydrate capping agent can comprise aplurality of oligosaccharides having a distribution of lengths. Thecarbohydrate capping agent can comprise one or more glucose residues.The carbohydrate capping agent can be in a substantially non-oxidizedform. In a refinement, reduction of the metal ion is performed in thepresence of a monosaccharide and/or a disaccharide in addition to thecarbohydrate capping agent. In another refinement, the carbohydratecapping agent comprises at least one of a monosaccharide and adisaccharide; and reduction of the metal ion is performed in thepresence of at least one non-carbohydrate capping agent in addition tothe carbohydrate capping agent. The carbohydrate capping agent can havea concentration in the aqueous medium selected to control one or moresize parameters of the plurality of metal nanoparticles formed duringreduction. The plurality of reduced metal nanoparticles can have anaverage particle size ranging from 2 nm to 50 nm, and it can have a(substantially) normal size distribution with a standard deviation of25% or less relative to the average particle size of the distribution.In an embodiment, at least some of the carbohydrate capping agent ispresent as a layer on an outer surface of each stabilized metalnanoparticle. In a refinement, the nanoparticle formation process canfurther include functionalization of the nanoparticles, for examplewherein (i) the aqueous medium in part (b) further comprises a bindingpair member comprising (A) an immobilization moiety for immobilizing thebinding pair member onto the reduced metal nanoparticle and (B) abinding moiety capable of binding to a target analyte or a secondbinding pair member; and (ii) part (b) is performed for a timesufficient in the presence of the binding pair member to additionallyimmobilize the binding pair member on an outer surface of the reducedmetal nanoparticle via the immobilization moiety. The immobilizationmoiety of the binding pair member can comprise a carbohydrate moietyconjugated to the binding moiety. In an embodiment, the reaction mediaand/or resulting compositions are free or substantially free (e.g., notmeasurable or not added to the reaction/product mixtures) ofnon-carbohydrate capping agents (e.g., citric acid or derivativesthereof such as citric acid (alkali metal) salts).

Additional features of the disclosure may become apparent to thoseskilled in the art from a review of the following detailed description,taken in conjunction with the drawings, examples, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the disclosure, reference should bemade to the following detailed description and accompanying drawingswherein:

FIGS. 1a-1d illustrate the AuNP synthesis process according to thedisclosure as in Example 1 and produced at optimal synthesis conditions:50° C., 24 hr, and 10.0 g/L dextrin; (a) TEM image of AuNP sample (scalebar: 200 nm); (b) High magnification of image (a) (scale bar: 50 nm);(c) UV-Vis spectra v. reaction time showing an increase with time of theAuNP characteristic absorbance peak (520 nm); and (d) visual colorchange reaction time series. The samples were diluted 1:4 with distilledwater for imaging.

FIGS. 2a-2c are TEM images of AuNPs generated at 50° C. during 24 hours.Insets show the size distribution for each sample. Dextrinconcentrations were (a) 20.0 g/L, (b) 10.0 g/L and (c) 2.5 g/L (scalebars for all three TEM images: 100 nm). FIG. 2d illustrates the averagefinal AuNP size as a function of initial dextrin concentration.

FIG. 3 is a graph illustrating AuNP particle size versus pH and dextrinconcentration.

FIG. 4 is a graph illustrating AuNP particle size versus dextrinconcentration after 24 hours at two generation temperatures.

FIG. 5 is a graph illustrating comparative capping efficiencies(percentage of captured fluorescence) of 12.4 nm and 10.4 nm dextrinAuNPs versus 13.0 nm citrate AuNPs. The observed capping efficiency of12.4 nm dextrin coated AuNPs and 13.0 nm citrate AuNPs was the same(left). A lower capping efficiency was observed when the dextrin AuNPs(10.4 nm) were smaller than the citrate AuNPs (13.0 nm) (right). Graphlegend: D=dextrin capping agent; C=citrate capping agent; * and ^indicate separate trials.

FIG. 6 is a graph illustrating the FTIR analysis of the dextrin cappingagent at different points in the synthetic process: (a) stock dextrin asreceived from manufacturer; (b) autoclaved dextrin solution at 20.0 g/L;and (c) recovered capping dextrin after particle generation.

FIG. 7 is a schematic of a target DNA detection system according toExample 2: (A) formation of complex target sandwich (MP-targetDNA-AuNP); (B) magnetic separation, metallic tracer (Au³⁺) dissolutionand electrochemical detection.

FIG. 8 is an agarose gel electrophoresis of tHDA products for thespecific isothermal amplification of a IS6110 fragment using individualreactants (Lane 1: 100 bp ladder; Lanes 2 and 3: tHDA amplificationfragment using 5 ng of synthetic target (190 bp); Lane 4: Blank).

FIGS. 9a and 9b are graphs illustrating the concentration-dependentresponse of the biosensor system of Example 2. (A) Mean DPV response ofsandwich complex (MNPs-target DNA-AuNPs) after hybridization fordifferent DNA target concentrations on SPCE. Each concentration was runin triplicates. The gold reduction peak was observed between 0.30-0.35 VDPV scan from +1.25V to 0V, step potential at 10 mV, modulationamplitude at 50 mV, and scan rate at 50 mV/S. (B) Logarithmiccorrelation between the target DNA concentration (ng/μl) and the goldreduction peak for three different trials. Each concentration was run intriplicates. The displayed equation corresponds to trial 1, which is thesame trial represented in the DPV response plot (A).

FIG. 10 is a TEM image of dextrin-capped, gold-coated magneticnanoparticles according to the disclosure (scale bar: 50 nm).

FIG. 11 illustrates a process for capturing and isolating/concentratingan analyte from a sample matrix using carbohydrate-capped, metal-coatedmagnetic nanoparticles according to the disclosure.

FIG. 12 is a graph presenting an electrochemical comparison in thedifferential current measured as a function of applied voltage formagnetic nanoparticles coated with either dextrin or citrate.

FIG. 13 is a graph confirming the functionalization of dextrin-capped,gold-coated magnetic nanoparticles using a fluorescence-labeled antibody(relative fluorescence units (RFU) of bound/immobilized antibody as afunction of antibody concentration).

FIG. 14 is a graph illustrating the capture efficiency ofantibody-functionalized, dextrin-capped, gold-coated magneticnanoparticles with respect to an E. coli O157:H7 bacterial targetanalyte extracted from a broth sample matrix as a function of analyteconcentration.

FIG. 15 is a schematic illustrating the capture, separation, anddetection of a target pathogen using a carbohydrate-capped metalnanoparticle according to the disclosure.

FIG. 16 is a graph comparing the differential pulse voltammetric (DPV)sensorgrams of dextrin-capped gold nanoparticles (AuNPs) and magneticnanoparticles (MNPs). The AuNPs show a characteristic current peak at0.25 V and the MNPs show a current peak at 0.58V.

FIG. 17 is a graph comparing the DPV sensorgram of AuNP-labeled E. coliO157:H7 detected with an SPCE biosensor at variable bacterial cellconcentrations (A: negative control, B: 10⁴ cfu/ml, C: 10⁶ cfu/ml, D:10² cfu/ml). The peak current for AuNP at about 0.25V to 0.3V increaseswith increasing cell concentration.

FIG. 18 is a graph of peak DPV current vs. cell concentration of theAuNP-labeled E. coli O157:H7. The signal shows a linear relationshipbetween 10¹ to 10⁶ cfu/ml.

FIG. 19 is a schematic illustrating the capture, separation, anddetection of a target pathogen using a metal nanoparticle including ananotracer (NT) according to the disclosure.

While the disclosed compositions, methods, and kits are susceptible ofembodiments in various forms, specific embodiments of the disclosure areillustrated in the drawings (and will hereafter be described) with theunderstanding that the disclosure is intended to be illustrative, and isnot intended to limit the claims to the specific embodiments describedand illustrated herein.

DETAILED DESCRIPTION

The disclosure generally relates to metal nanoparticle compositions andtheir methods of formation, in particular gold nanoparticles (AuNPs)such as solid AuNPs or nanoparticles with a gold (shell)-nanoparticle(core) structure. The metal nanoparticles can be in the form of a metalnanoparticle core stabilized by the carbohydrate capping agent (e.g., ametal nanoparticle formed substantially entirely from gold).Alternatively, the metal nanoparticles can be in the form of ananoparticle core (e.g., non-metallic and/or magnetic) having a metalcoating in a core-shell configuration (e.g., a magnetic iron oxide-goldcomposite particle in a core-shell configuration), where the core-shellnanoparticle is stabilized by the carbohydrate capping agent (e.g., viainteractions between the metal shell and the capping agent).Compositions according to the disclosure include aqueous suspensions ofmetal nanoparticles that are stabilized with one or more carbohydratecapping agents. The nanoparticle suspensions are stable for extendedperiods (e.g., for at least several months) and can be functionalized asdesired at a later point in time, typically prior to use in an assay forthe detection of a target biological analyte (e.g., using an antibody,DNA probe, or other biomolecular probe capable of binding to the targetanalyte as a functionalization agent). The stable nanoparticlesuspension can be formed by the aqueous reduction of metal precursorions at non-acidic pH values in the presence of a carbohydrate-basedcapping agent such as dextrin or other oligosaccharides. In someembodiments, functionalization of the metal nanoparticles can beperformed in the same reaction system as that used for nanoparticleformation, for example simultaneously performed with some or all of thenanoparticle formation process.

Metal Nanoparticle Formation

Methods of metal nanoparticle formation according to the disclosuregenerally are performed in an aqueous reaction system including metalions to be reduced in solution in the aqueous medium. The metal ions inthe aqueous medium are reduced at a neutral or alkaline pH value in thepresence of a carbohydrate capping agent under suitable reactionconditions to form a plurality of reduced metal nanoparticles (e.g., ata reaction temperature and reaction time sufficient to convert all orsubstantially all of the metal ion precursors). The reaction generallyincludes an initial nucleation stage to form metallic nuclei followed bya longer growth stage in which metal ions reduced on the nuclei surfacescreate the final metal nanoparticles. The plurality of reduced metalnanoparticles are in the form of a stabilized suspension of metalnanoparticles in the aqueous medium, where the carbohydrate cappingagent stabilizes the formed nanoparticle suspension.

The specific metal ions or oxidized metal-containing species in solutionand selected as precursors to the desired metal nanoparticles are notparticularly limited and are suitably chosen according to a desired enduse/application of the nanoparticle suspension. In an embodiment, themetal ions include gold ions (e.g., Au(III), Au³⁺) and are selected toform gold metal nanoparticles (AuNPs). The metal ions can be free insolution or coordinated/coupled with other (ionic) species (e.g., Au³⁺,[AuCl₄]⁻, [AuCl₃OH]⁻, [AuCl₂(OH)₂]⁻, [AuCl(OH)₃]⁻ or [Au(OH)₄]⁻, wherethe oxidation level of gold in each case is +3). Other potential metalions can include chromium, copper, zinc, nickel, cadmium, silver,cobalt, indium, germanium, tin, lead, arsenic, antimony, bismuth,chromium, molybdenum, manganese, iron, ruthenium, rhodium, palladium,osmium, iridium, and platinum. In some embodiments, two or more types ofmetal ions can be in solution in the aqueous medium to provide metalnanoparticles formed from alloys of two or more elemental metals. Theconcentration of metal ions in solution prior to reaction is notparticularly limited, but it suitably ranges from 0.1 mM to 1000 mM(e.g., at least 0.1 mM, 1 mM, or 10 mM and/or up to 100 mM or 1000 mM).

The metal ions are suitably introduced into the aqueous medium as adissolvable ionic compound, for example a salt or acid. A suitablesource of gold ions is chloroauric acid (HAuCl₄), which can provideAu(III) in the form of [AuCl₄]⁻. Other salts/compounds including theoxidized metal precursor such as halides (e.g., chlorides, bromides,fluorides, iodides), sulfates, sulfites, thiosulfates, nitrates,nitrites, carboxylates, sulfonates, and hydrogenated forms thereof(e.g., as in HAuCl₄) can be used as desired and depending on theparticular metal ion to be introduced into the aqueous medium.

In some embodiments, the aqueous medium further includes, prior toreduction of the metal ions, a population of nanoparticles serving ascores/nucleation sites for deposition of the reduced metal ions, thuspermitting the formation of metal nanoparticles having a core-shellstructure including a nanoparticle core with a metallic shell. Thenanoparticle core material is not particularly limited and can benon-metallic, metallic (e.g., different from the metal to be reduced asa shell), magnetic, etc. Magnetic nanoparticle cores are particularlyuseful to permit the resulting metal nanoparticle to function as both amagnetic sample/analyte separator and concentrator (e.g., due to themagnetic core) as well as a signal transducer (e.g., due to theelectrical properties of the metal shell material such as gold).

The magnetic nanoparticles according to the disclosure are notparticularly limited and generally include any nano-sized particles(e.g., about 1 nm to about 1000 nm) that can be magnetized with anexternal magnetic/electrical field. The magnetic nanoparticles moreparticularly include superparamagnetic particles, which particles can beeasily magnetized with an external magnetic field (e.g., to facilitateseparation or concentration of the particles from the bulk of a samplemedium) and then redispersed immediately once the magnet is removed(e.g., in a new (concentrated) sample medium). Thus, the magneticnanoparticles are generally separable from solution with a conventionalmagnet. Suitable magnetic nanoparticles are provided as magnetic fluidsor ferrofluids, and mainly include nano-sized iron oxide particles(Fe₃O₄ (magnetite) or γ-Fe₂O₃ (maghemite)) suspended in a carrierliquid. Such magnetic nanoparticles can be prepared by superparamagneticiron oxide by precipitation of ferric and ferrous salts in the presenceof sodium hydroxide and subsequent washing with water. A suitable sourceof γ-Fe₂O₃ is Sigma-Aldrich (St. Louis, Mo.), which is available as anano-powder having particles sized at <50 nm with a specific surfacearea ranging from about 50 m²/g to about 250 m²/g. Preferably, themagnetic nanoparticles have a small size distribution (e.g., rangingfrom about 5 nm to about 25 nm) and uniform surface properties (e.g.,about 50 m²/g to about 245 m²/g).

More generally, the magnetic nanoparticles can include ferromagneticnanoparticles (i.e., iron-containing particles providing electricalconduction or resistance). Suitable ferromagnetic nanoparticles includeiron-containing magnetic metal oxides, for example those including ironeither as Fe(II), Fe(III), or a mixture of Fe(II)/Fe(III). Non-limitingexamples of such oxides include FeO, γ-Fe₂O₃ (maghemite), and Fe₃O₄(magnetite). The magnetic nanoparticles can also be a mixed metal oxideof the type M1_(x)M2_(3-x)O₄, wherein M1 represents a divalent metal ionand M2 represents a trivalent metal ion. For example, the magneticnanoparticles may be magnetic ferrites of the formula M1Fe₂O₄, whereinM1 represents a divalent ion selected from Mn, Co, Ni, Cu, Zn, or Ba,pure or in admixture with each other or in admixture with ferrous ions.Other metal oxides include aluminum oxide, chromium oxide, copper oxide,manganese oxide, lead oxide, tin oxide, titanium oxide, zinc oxide andzirconium oxide, and suitable metals include Fe, Cr, Ni or magneticalloys.

Reduction of the metal ions in the aqueous medium is performed at aneutral or alkaline pH value, for example ranging from 7 to 12 (e.g.,where the pH value is essentially constant throughout the reaction, orit may vary within the range during reaction). In various embodiments,the pH value of the reaction medium can be at least 7, 7.5, 8, 8.5, 9and/or up to 8, 8.5, 9, 9.5, 10, 11, 12. The selection and control ofthe desired pH value can be effected by any suitable base and/or buffersystem as is generally know in the art. As described below, in someembodiments, the pH value can be controlled by selection of a reducingagent. Non-acidic pH values, in particular those that are mildly basicor otherwise near to a physiological pH value, are desirable in certainembodiments to promote functionalization of the eventual metalnanoparticles with biomolecules that would be denatured or whoseactivity would otherwise be reduced or negated in an acidic environment.

The reaction temperature of the reduction process is not particularlylimited, for example being at room temperature (e.g., 20° C. to 25° C.)or at mildly elevated temperatures relative to room temperature. Invarious embodiments, the temperature of the aqueous medium can rangefrom 20° C. to 100° C. during the reduction reaction, for example beingat least 20° C., 25° C., 30° C., 35° C., or 40° C. and/or up to 30° C.,35° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C., or 100° C. invarious embodiments.

Reduction of the metal ions in the aqueous medium is suitably effectedby the addition of a chemical reducing agent to the aqueous medium.Suitable reducing agents are those that are effective at reducingmetallic ions at the neutral/alkaline pH of the aqueous medium (e.g.,they do not require an acidic pH and/or do not themselves create anacidic environment). In some embodiments, the reducing agent is acombined reducing agent for reducing the metal ions and pH-adjustingagent for maintaining the neutral or alkaline pH value of the aqueousmedium. Suitable combined reducing and pH-adjusting agents include metal(e.g., alkali or alkali earth metal) carbonates or bicarbonates such assodium carbonate (Na₂CO₃). However, other reducing agents that areoperative at neutral/alkaline pH values can be used even if they do notalso function as a pH-adjusting agent (e.g., in which case othernon-reducing bases/buffers can be used to independently control the pHvalue). Examples of other suitable reducing agents include hydrides(e.g., lithium aluminum hydride (LiAlH₄), sodium borohydride (NaBH₄),diisobutylaluminum hydride (DIBAH)), dithiothreitol (DTT),sulfites/bisulfites (e.g., ammonium, metallic such as from alkali andalkali earth metals including K, Na, Li, Mg, Ba, Ca), sulfates (e.g.,metallic such as from iron (II) or other soluble iron (II) salts),peroxides (e.g., those functioning as reducing agents at alkaline pHsuch as hydrogen peroxide (H₂O₂)), sulfides (e.g., metallic such as fromalkali metals like Na), and amines (e.g., including ammonium saltsthereof such as hydroxylamine (NH₂OH) or hydroxylamine hydrochloride(NH₂OH.HCl)).

The carbohydrate useful as a capping agent according to the disclosureis generally an oligo- or polysaccharide having a plurality ofsaccharide residues (e.g., having a general formula C_(m)(H₂O)_(n) forunmodified carbohydrates with residues derived from monosaccharideshaving a general formula (CH₂O)_(n)). In some embodiments, thecarbohydrate capping agent can be a carbohydrate derivative, for examplehaving additional functional groups such as carboxylate group ornitrogen-containing groups (e.g., amino, N-acetyl). The capping agentcan include linear and/or branched carbohydrates, such as thoseincluding α- or β-glycosidic bonds (e.g., α(1,4) or α(1,6) glycosidiclinkages as in dextrin or other starch-based capping agents). Thespecific carbohydrate capping agent is suitably selected so that it hasat least some hydrophilic character (e.g., to promote a water-stablesuspension), and it can be a water-soluble carbohydrate in somerefinements. In some embodiments, the capping agent is in asubstantially non-oxidized form (e.g., being (substantially) free fromaldose, ketose, and/or carboxylate (acid or anion) functionalitieseither for a portion of or the whole capping agent molecule; based on anabsence of such functionalities and/or the inability to detect(non-trace) levels of the functionalities in the capping agent), forexample as added to the reaction mixture, as present during reaction,and/or as bound/conjugated to the metal nanoparticles in the reactionproduct. In other embodiments, other non-carbohydrate capping agentssuch as polyethylethene glycol (e.g., or other polyether or polyethyleneoxide), various silanes, polyacrylamide, and other negatively chargedpolymers can be used (e.g., for use instead of or in combination withother carbohydrate capping agent such as oligosaccharide; suitably incombination with a monosaccharide, a disaccharide, or a derivativethereof as described below as an additive to the carbohydrate cappingagent system). The concentration of the capping agent in solution priorto reaction is not particularly limited, but it suitably ranges from 1,2, 5, or 10 g/L to 15, 25, 35, 50, or 100 g/L (e.g., where selection ofthe capping agent concentration can permit selection of an average metalnanoparticle size and/or size distribution resulting from theconcentration).

The capping agent is suitably an oligosaccharide having 3 to 100saccharide residues, for example at least 3, 5, 10, 15, 20, 25, 30, or40 and/or up to 10, 20, 30, 40, 50, 60, 80, or 100 saccharide residues.In some embodiments, the capping agent represents a plurality ofoligosaccharides or polysaccharides having a distribution ofsizes/lengths (e.g., in terms of number of saccharide residues). In suchcases, ranges characterizing the oligosaccharide capping agent in termsof number of saccharide residues can represent an average of thedistribution (e.g., number or other average), or the ranges canrepresent upper and lower bounds for the distribution (e.g., within 1,2, or 3 standards deviations from the mean; representing the 1%/99%,5%/95%, or 10%/90% cut points of the cumulative size distribution).

In some embodiments, the carbohydrate capping agent can include one ormore glucose residues (e.g., D-glucose; having a plurality of glucoseresidues such as where the capping agent essentially consists only ofglucose residues). However, the capping agent can include othersaccharide residues alone, in combination with glucose, and/or incombination with each other, for example including those from allose,altrose, mannose, gulose, iodose, galactose, talose, xylose, arabinose,fucose, and/or fructose. As noted above, the capping agent can includecarbohydrate derivates, for example including saccharide residues fromglucuronic acid (e.g., also including salts and esters thereof),N-acetyl-D-glucosamine (e.g., derived from chitin), and D-glucosamine(e.g., derived from chitosan).

Oligomeric carbohydrate capping agents containing the various saccharideresidues can be (synthetic) oligosaccharides having a selectedlength/saccharide sequence, or they can be formed from naturallyoccurring polysaccharides. Polysaccharides can be subjected to enzymaticor other chemical forms of hydrolysis to form shorter oligosaccharides,generally with an element of random size distribution. Examples ofsuitable precursor polysaccharides for capping agents include starch(e.g., forming dextrin), amylose, amylopectin, cellulose, laminarin,chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, andgalactomannan. In an embodiment, the capping agent is a dextrin (e.g.,linear, branched, or cyclic; suitably linear and/or branched having atleast 10, 20, or 30 saccharide residues), for example being formed fromstarch (e.g., including amylose and/or amylopectin).

In an embodiment, the aqueous medium can include a saccharide-basedmoiety in addition to the carbohydrate capping agent during metal ionreduction. The additional saccharide-based moiety can be included toform metal nanoparticle suspensions that remain stably suspended foreven longer periods (i.e., in comparison to suspensions stabilized withthe capping agent alone) and can be a reducing sugar. The additionalstabilizing agent is generally a monosaccharide, a disaccharide, or aderivative thereof. Suitable examples include sucrose, glucose,fructose, mannose, galactose, glyceradehyde, lactose, and maltose,although the additional stabilizing agent more generally can include anycombination of the saccharide residues listed above for theoligomeric/carbohydrate capping agent.

Stabilized Metal Nanoparticle Compositions

The above process results in the formation of a metal nanoparticlecomposition. Once the reduction reaction has progressed (e.g., tocompletion, such as once substantially all precursor metal ion reactanthas been consumed), the aqueous medium contains a plurality of reducedmetal nanoparticles as a suspension stabilized in the aqueous mediumwith the carbohydrate capping agent. Accordingly, the disclosure alsorelates to a stabilized metal nanoparticle suspension composition thatincludes water in a sufficient amount to provide an aqueous medium andstabilized metal nanoparticles stably suspended in the aqueous medium.The aqueous medium suspension can have the same neutral or alkaline pHas that used for metal ion reduction (e.g., ranging from 7 to 12), or itcan be adjusted to a different pH value post-reduction (e.g., stillgenerally in the neutral or alkaline range) for storage or to facilitatesubsequent functionalization. The stabilized metal nanoparticles in thesuspension individually can include a metal nanoparticle core (e.g.,generally having a spherical or nearly spherical/spheroidal shape) and acarbohydrate capping agent present as a layer on an outer surface of themetal nanoparticle core in an amount sufficient to stabilize the metalnanoparticle suspension (i.e., the capping agent need not completelyenvelop the nanoparticle core, but it is present near the core surfacein a sufficient amount to prevent/inhibit substantial settling oragglomeration of the nanoparticles). Similarly, stabilized metalnanoparticles in the suspension individually can include a core-shellnanoparticle and a carbohydrate capping agent present as a layer on anouter surface of the metal nanoparticle shell in an amount sufficient tostabilize the metal nanoparticle suspension. In various embodiments, thecarbohydrate capping agent can form a complete or partial layer (e.g., amonolayer or a plurality of layers) that is adsorbed or otherwise boundto the metal nanoparticle surface such as by electrostatic interactionsbetween the metal nanoparticle surface and hydroxyl groups of thecarbohydrate capping agent present at the neutral or alkaline pH of theaqueous medium.

The population of the reduced metal nanoparticles as produced (e.g., insuspension as formed in the aqueous medium or otherwise) generally has aparticle size ranging from 2 nm to 50 nm (e.g., a number-, weight-, orvolume-average particle size). For example, the average size of thenanoparticle distribution can be at least 2, 5, 8, 10, 12, or nm and/orup to 8, 10, 12, 15, 20, 25, 30, 40, or 50 nm. In an embodiment, thedistribution of metal nanoparticles also has a relatively narrow sizedistribution, for example a substantially normal size distribution witha standard deviation of 25% or less relative to the average particlesize of the distribution (e.g., a monomodal distribution; having a σ/<x>for a normal distribution of not more than 25%, 20%, 15%, or 10% and/orat least 1%, 2%, 5%, 8% or 10%). As illustrated in the examples below,various size parameters of the metal nanoparticle distribution (e.g.,average size, distribution width) can be selected/controlled byselecting one or more reduction reaction parameters. Examples ofsuitable reaction/operating conditions that can be selected to controlnanoparticle size include capping agent concentration, metal ionconcentration, reducing agent concentration, reaction temperature,reaction pH, length and/or size distribution of the oligomeric cappingagent.

The capping agent-stabilized metal nanoparticles remain stably suspendedin the aqueous medium for extended periods without (substantial)settling or agglomeration of the nanoparticles. For example, thesuspension can remain stable for at least 90 days when stored at roomtemperature. In various embodiments, the suspension is stable or capableof remaining stable for periods of at least 90, 120, 180, 270, or 360days and/or up to 270, 360, 720, or 1080 days and/or at storagetemperatures generally between 20° C. and 25° C., in particular at aneutral or alkaline pH. The metal nanoparticles remain stably suspendedin the aqueous medium in part based on the hydrophilic character ofvarious functional groups the carbohydrate capping agent (e.g., hydroxylgroups, which can impart a water-soluble character to low-molecularweight capping agents).

Functionalization of Metal Nanoparticles

The carbohydrate-capped metal nanoparticles can be functionalized, forexample with a biomolecule, according to various methods known in theart for any desired nanoparticle application (e.g., use in a biosensorto detect a target biological analyte, use as a vehicle for deliveringthe functionalized biomolecule to a target). General methods ofbiomolecule attachment can include physical adsorption (e.g., resultingfrom electrostatic metal-biomolecule interactions), direct binding(e.g., based on affinity interactions between the metal and a functionalgroup of the biomolecule, such as between a thiolated biomolecule andgold), covalent attachment (e.g., between the biomolecule and a covalentlinking intermediate that is bound to the metal nanoparticle, such asthrough thiolated carboxylic acids, EDAC-mediated attachment ofbiomolecules, biotin-streptavidin linking, and azide-linking or other“click” functionalization techniques) (DeLong 2010).

Functionalization of the metal nanoparticles is generally performed byimmobilizing a binding pair member on the metal nanoparticles (e.g., onthe outer nanoparticle surface). The binding pair member can be selectedfor its ability to specifically or non-specifically bind to a targetanalyte (e.g., a protein, virus, bacteria, ssDNA, such a DNA of a targetmicroorganism or complementary ssDNA when the binding pair member is forimmobilization of a biobarcode) or for its ability to form covalentbonds or otherwise bind with a second binding pair member (e.g., thatitself can specifically or non-specifically bind to a target analyte).

The binding pair member includes an immobilization moiety forimmobilizing the binding pair member onto the metal nanoparticle and abinding moiety. In some embodiments, the immobilization moiety is afunctional group that is normally part of the binding pair member (e.g.,a polar group capable of electrostatic interactions). In otherembodiments, the immobilization moiety is a functional group that isadded to the binding pair member to facilitate binding to the metalnanoparticle (e.g., a thiol group for gold attachment; acarbohydrate/saccharide moiety for enhanced electrostatic interactionswith a metal surface). In some embodiments, the binding moiety is theportion of the binding pair member that is capable of specific ornon-specific binding to the target analyte (e.g., ssDNA probe for ssDNAbinding/detection, binding region of antibody for virus/bacteriabinding/detection). In other embodiments, the binding moiety is theportion of binding pair member that is capable of binding to the secondbinding pair member (e.g., a carboxylate group such as a carboxylic acidor salt capable of forming covalent links to an immobilization moiety ofthe second binding pair member).

In one embodiment, the binding pair member can be immobilized on themetal nanoparticle using a ligand exchange process known in the art. Ina general ligand exchange process, the carbohydrate capping agentstabilizing the metal nanoparticle suspension is removed from the outersurface of the metal nanoparticles (e.g., partial or complete removal ofthe capping agent). Removal of the capping agent promotes increasedaccess to surface areas of the metal nanoparticles, thus allowingimmobilization of the binding pair member on the outer surface of themetal nanoparticle via the immobilization moiety (e.g., bycontacting/incubating the metal nanoparticle suspension with the bindingpair member). As illustrated in Examples 1 and 2 below, a suitableligand exchange method for gold nanoparticles includes a DTT-mediatedremoval of the carbohydrate capping agent followed by immobilization ofa thiolated binding pair member (e.g., thiolated ssDNA oligonucleotide)on the gold nanoparticle surface. As illustrated in Example 3 below,another suitable ligand exchange method for gold nanoparticles includesa surfactant-mediated removal of the carbohydrate capping agent followedby immobilization of a thiolated binding pair member (e.g., thiolatedcarboxylic acid used for further covalent attachment) on the goldnanoparticle surface.

In another embodiment, specific binding pair members such as antibodiescan be immobilized on the metal nanoparticle via adsorption. Forexample, antibodies can be bound (e.g., by direct physical adsorption)to the outer metal portion of the metal nanoparticle by incubating theantibodies in a buffer suspension of the metal nanoparticles.Additionally, an immunoglobulin (antibody) with an Fc region and anantigen-binding region capable of specifically binding to the targetanalyte (e.g., IgA, IgD, IgE, IgG, IgM, subclasses thereof, andcombinations thereof) can be incubated with an immunoglobulin-bindingprotein having a binding affinity to the Fc region of the immunoglobulin(e.g., a bacterial surface protein such as protein A, protein G, proteinA/G, and combinations thereof), such that the immunoglobulin-bindingprotein binds to the outer metal portion of the metal nanoparticle(e.g., via adsorption) and the Fc region of the immunoglobulin, therebypreferentially orienting the resulting immunoconjugate so that theantigen-binding region of the immunoglobulin is outwardly directedrelative to the metal nanoparticle (e.g., which enhances binding/captureefficiency of the immunoglobulin).

In another embodiment, the binding pair member can be immobilized on themetal nanoparticle in the same aqueous reaction medium used for metalion reduction and metal nanoparticle formation (e.g., a “one-pot”synthesis). In such cases, functionalization of the nanoparticles can beperformed during the reduction reaction (e.g., initiation offunctionalization step at the same time the reduction reaction isinitiated or at a subsequent time when the reduction reaction is stillproceeding), or functionalization can be performed subsequent to thereduction reaction (e.g., initiation of functionalization stepimmediately after or a short time after the reduction reaction iscomplete, such as within 24 hours). Addition and incubation of thebinding pair member in the aqueous medium for a sufficient time (e.g.,under suitable temperature and pH conditions, such as those suitable forthe reduction reaction itself) allow immobilization of the binding pairmember on the outer surface of the metal nanoparticle via theimmobilization moiety. In an embodiment, the binding pair member/bindingmoiety thereof can be conjugated to a carbohydrate moiety analogous toany of those described above in relation to the carbohydrate cappingagent (e.g., a monosaccharide, a disaccharide, and/or an oligosaccharideof the various saccharide residues or derivatives thereof). In onerefinement, the aqueous reduction medium can include carbohydratemoieties both from the capping agent (e.g., which is free from bindingmoieties or binding pair members) and from those conjugated to thebinding pair members. In another refinement, the only carbohydratemoieties present in the aqueous medium are capping agent species thatare also conjugated to the binding pair members (e.g., in which case thecarbohydrate serves as both the capping agent for nanoparticleformation/stabilization and the immobilization moiety for the bindingpair member). Methods of conjugating saccharide moieties to moleculesuseful as binding pair members (e.g., ssDNA oligonucleotides) are knownin the art (Adinolfi 2004; Pourceau 2009).

Binding Pair Members

As described above, the metal nanoparticles can be functionalized withbinding pair members to detect a variety of target analytes in biosensorapplications. The binding pair member is selected to be capable ofbinding (specific or non-specific) to a target analyte so that the metalnanoparticle composition can be used for the (selective) detection ofthe target analyte in a sample.

An analyte (or target analyte) generally includes a chemical orbiological material, including living cells, in a sample which is to bedetected using the functionalized nanoparticle composition or otheranalyte probe. The analyte can include pathogens of interest (e.g.,bacterial pathogens such as E. coli O157:H7, B. anthracis, B. cereus, inaddition to those listed above). The analyte also may be an antigen, anantibody, a ligand (i.e., an organic compound for which a receptornaturally exists or can be prepared, for example one that is mono- orpolyepitopic, antigenic, or haptenic), a single compound or plurality ofcompounds that share at least one common epitopic site, and a receptor(i.e., a compound capable of binding to an epitopic or determinant siteof a ligand, for example thyroxine binding globulin, antibodies,enzymes, Fab fragments, lectins, nucleic acids, protein A, complementcomponent C1q). In some embodiments, the term “analyte” also can includean analog of the analyte (i.e., a modified form of the analyte which cancompete with the analyte for a receptor) that can also be detected usingthe functionalized nanoparticle nanoparticle composition.

The specific binding pair member generally includes one of two differentmolecules, each having a region or area on its surface or in a cavitythat specifically binds to (i.e., is complementary with) a particularspatial and polar organization of the other molecule. The binding pairmembers can be referenced as a ligand/receptor (or antiligand) pair.These binding pair members include members of an immunological pair suchas antigen-antibody. Other specific binding pairs such as biotin-avidin(or derivatives thereof such as streptavidin or neutravidin),hormones-hormone receptors, IgG-protein A, polynucleotide pairs (e.g.,DNA-DNA, DNA-RNA), DNA aptamers, biomimetic antibody-antigen (e.g.,molecularly imprinted synthetic polymer having specific bindingcapability with the antigen), and whole cells are not immunologicalpairs, but can be used as binding pair members within the context of thepresent disclosure.

Preferably, the binding pair members are specific to each other and areselected such that one binding pair member is the target analyte ofinterest or a component thereof (e.g., a specific surface protein orother surface component of specific bacteria or other pathogen ofinterest), and the other binding pair member is the constituent bound tothe conductive polymer of the particulate composition. Bindingspecificity (or specific binding) refers to the substantial recognitionof a first molecule for a second molecule (i.e., the first and secondmembers of the binding pair), for example a polypeptide and a polyclonalor monoclonal antibody, an antibody fragment (e.g., a Fv, single chainFv, Fab′, or F(ab′)₂ fragment) specific for the polypeptide,enzyme-substrate interactions, and polynucleotide hybridizationinteractions. Preferably, the binding pair members exhibit a substantialdegree of binding specificity and do not exhibit a substantial amount ofnon-specific binding (i.e., non-covalent binding between molecules thatis relatively independent of the specific structures of the molecules,for example resulting from factors including electrostatic andhydrophobic interactions between molecules).

Substantial binding specificity refers to an amount of specific bindingor recognition between molecules in an assay mixture under particularassay conditions. Substantial binding specificity relates to the extentthat the first and second members of the binding pair to bind only witheach other and do not bind to other interfering molecules that may bepresent in the analytical sample. The specificity of the first andsecond binding pair members for each other as compared to potentialinterfering molecules should be sufficient to allow a meaningful assayto be conducted for the target analyte. The substantial bindingspecificity can be a function of a particular set of assay conditions,which includes the relative concentrations of the molecules, the timeand temperature of an incubation, etc. For example, the reactivity ofone binding pair member with an interfering molecule as compared to thatwith the second binding pair member is preferably less than about 25%,more preferably less than about 10% or about 5%.

A preferred binding pair member is an antibody (an immunoglobulin) thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule (e.g., anantigen). Antibodies generally include Y-shaped proteins on the surfaceof B cells that specifically bind to antigens such as bacteria, viruses,etc. The antibody can be monoclonal or polyclonal and can be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal) or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal), or bycloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies may include acomplete immunoglobulin or fragment thereof, which immunoglobulinsinclude the various classes and isotypes, such as IgA, IgD, IgE, IgG1,IgG2a, IgG2b, IgG3, IgM, etc. Fragments thereof may include Fab, Fv andF(ab′)₂, and Fab′. In addition, aggregates, polymers, and conjugates ofimmunoglobulins or their fragments can be used where appropriate so longas binding affinity for a particular molecule is maintained.

EXAMPLES

The following examples illustrate the disclosed compositions andmethods, but are not intended to limit the scope of any claims thereto.

Example 1 Synthesis of Dextrin-Capped Gold Nanoparticles

The synthesis described in this example generates glyco-AuNPs under mildalkaline conditions in an aqueous medium to provide a “greener”alternative to Brust and Turkevich methodologies. The biologicallycompatible, one-step technique in this example used dextrin as a cappingagent and sodium carbonate as a reducing agent for a chloroauric acidgold precursor. The generated particles were relatively mono-dispersedand water soluble with a range of controllable mean diameters from 5.9to 16.8 nm±1.6 nm. The produced AuNPs were stable in water for more thansix months stored at room temperature (21° C.) in the generationsolution and without protection from light. The example furtherevaluates the effect of temperature, pH, and dextrin concentration onthe synthesis procedure and the resulting AuNP diameter of the AuNPdistribution. These factors were found to control the reaction speed.The produced glyco-AuNPs were successfully functionalized with DNAoligonucleotides, and the functionalization efficiency was similar tocitrate-generated AuNPs. The alkaline synthesis potentially allowssimultaneous synthesis and functionalization procedures, which couldsignificantly reduce the time of current ligand exchange methodologies.

AuNP Synthesis:

A gold chloride (HAuCl₄) stock solution (20 mM) (#520918-5G fromAldrich) was prepared using distilled sterile water and was stored underrefrigeration. The dextrin stock solution (25 g/L) (#31400 Fluka) wasprepared using deionized water and autoclaved prior to use. A mixture ofdistilled sterile water and dextrin stock solution was added to asterile 250 mL flask according to the desired dextrin concentrationwithin the 2.5 to 20.0 g/L final working range. A volume of 5 mL ofHAuCl₄ stock solution was added to the reaction and adjusted to pH 9using filter-sterile 10% sodium carbonate (Na₂CO₃), the final HAuCl₄concentration in the reaction was 2 mM. Finally, the reaction volume (50mL) was completed by using pH adjusted distilled water (pH 9). The flaskwas incubated in the dark at 50° C. with continuous shaking for 8 hours.Particle formation was observed through the following stages of colorchange; clear, purple tint, red tint, and red (520 nm), the same colorsequence as citrate reduction (Polte et al. 2010). The effects of pH(from 3 to 11) and temperature (25° C. and 50° C.) on the synthesizedparticles were evaluated and compared to sodium citrate generated AuNPs(Hill and Mirkin 2006).

AuNP Characterization:

The AuNP formation was evaluated using UV/Vis scanning spectroscopy, andthe size distribution by light scattering and transmission electronmicroscopy (TEM). UV/Vis spectra were measured using a UV-VIS-NIRScanning Spectrophotometer (Shimadzu). Particle size and distributionwere obtained from TEM images and collected with a JEOL 100CXTransmission Electron Microscope. A 1:4 diluted suspension of the AuNPsin distilled water was sonicated for 5 min before 5 μl of the sample wastransferred to a formvar/carbon coated copper grid (300 mesh) forimaging.

AuNP Functionalization:

In order to evaluate the ability to use the dextrin-coated AuNPs forfuture sensing assays, thiol-modified oligonucleotides were attached tothe AuNP surface using the ligand exchange method for functionalizingcitrate-coated AuNPs (Hill and Mirkin 2006; Zhang et al. 2009). Anoligonucleotide (sequence 5′-TTA TTC GTA GCT AAA AAA AAA A-3′; SEQ IDNO: 1) was used with 5′ 6-Carboxyfluorescein (6-FAM; λ excitation=495nm, λ emission=520 nm) and 3′ thiol modifications (IDT Coralville,Iowa). The oligonucleotide was ligand-exchanged for the capping agentpost-AuNP production. The AuNPs were centrifuged into a pellet andseparated from the excess DNA ligand, washed 3 times, and resuspended indithiothreitol (DTT) buffer. The DTT AuNP-DNA solution was heated for 60minutes at 50° C. in order to ligand-exchange DTT for the thiolated DNA.The fluorescence signal from the supernatant was measured for DNAconjugation efficiency using a 527-547 nm emission filter (VICTOR3 1420Multilabel counter, Perkin-Elmer).

Results—AuNP Synthesis:

AuNPs were successfully synthesized at alkaline conditions using dextrinas a capping agent. FIG. 1a shows a typical TEM image generated at 10.0g/L dextrin concentration, 2 mM HAuCl₄, pH 9.0 and 50° C. The particlegeneration was monitored over 24 hours with UV/Vis specta. Theabsorbance peak at 520 nm was observed in all samples after initial redtint through the characteristic wine-red color of gold nanoparticles inthe 10-100 nm size range. The AuNP formation was visually observed after6 hours of incubation. The reaction continued until completion at 8hours and monitored over the course of 24 hours with TEM images (FIGS.1a and 1b ). FIG. 1c shows the increase of absorbance at 520 nm withtime and the corresponding change in color can be observed in FIG. 1d .Particle formation was not observed when dextrin, pH adjustment, orsodium carbonate was excluded from the synthesis.

Effect of Dextrin Concentration:

The ratio of capping agent to particle concentration is a commonlyvaried factor to control the final size of the particles. Theconcentration of dextrin was varied from 2.5 g/L to 20.0 g/L to explorethe effect of dextrin concentration on particle size. The final size ofparticles was determined using TEM images (FIGS. 2a-2c ). It wasobserved that the particle diameter decreased with increasing dextrinconcentration. Particle sizes were 8.6 nm±1.2 nm, 10.6 nm±1.6 nm, and12.4 nm±1.5 nm (expressed as a mean+/−one standard deviation of a normaldistribution) for 20.0, 10.0, and 2.5 g/L dextrin concentrations,respectively. FIG. 2d shows a linear relationship between dextrinconcentration and particle size for 2.5 to 20.0 g/L of dextrin.

The initial yellow color from the HAuCl₄ solution changes to clear withthe addition of sodium carbonate for pH adjustment. The time required tochange from colorless to red decreased with increased initial dextrinconcentration. Initial particle formation was observed within 5 hours at2.5 g/L of dextrin, and within 1.5 hours for 20.0 g/L of dextrinindicated by the wine red color, which became more intense as thereaction completed. UV-Vis spectral data (not shown) were used tomonitor the reactions for completion. All dextrin concentrations between5.0 and 20.0 g/L showed completion at 6 hours, with 2.5 g/L showingcompletion at 24 hours.

The size and production rate appear to be controlled by dextrinconcentration. Polte et al. (2010) proposed a four step generation modelfor citrate-coated AuNPs. Without wishing to be bound by any particulartheory, it appears that alkaline dextrin system presented here appearsto follow a similar mechanism, that is, from (1) reduction, (2)stabilization, (3) exchange (slow growth phase), and finally to (4)capping (fast growth phase). Steps 1 and 2 could be carried out by thesodium carbonate reducing a form of [AuCl₄]⁻, [AuCl₃OH]⁻, [AuCl₂(OH)₂]⁻,[AuCl(OH)₃]⁻ or [Au(OH)₄]⁻ to Au⁰, the degree of hydroxyl coordinationbeing dependent on pH. In the early steps, the oxidized carbonate wouldbe stabilizing the initial AuNP instead of the citrate molecule. As thereaction proceeds into the slow growth phase, step 2-3, the exchange ofcarbonate for dextrin occurs which explains the extended time it takesfor the growth phase to complete, as observed in the transition from thepurple tint and the initial red color. Once the system enters step 4,fast growth, dextrin is believed to be the sole capping agent and thedisassociation allows for auto-catalytic growth and rapid re-associationof free dextrin molecules. This growth stage in the alkaline generationmethodology is estimated to be the 30-60 minute time window for thepurple tint to transition to the wine red color. Changes in dextrinconcentration for this system may explain why the rate increased and thesize decreased with higher concentration. The smaller particle sizesresult when higher amount of capping agents are used. A greater cappingagent concentration can cover greater surface areas and generate smallersized particles (Wang and Yang 2006). The increased rate of generationmay be explained by either bulk interaction or association. In a higherconcentration system, dextrin may interact with the carbonate on thegold surface promoting faster carbonate disassociation. As the carbonatedisassociates, greater concentrations of free dextrin undergo surfacecapping more rapidly.

Effect of pH:

Recent reports of sodium hydroxide induced particle formation (Zhou etal. 2009) suggest that hydroxyl ions can reduce Au³⁺ into Au⁰, but donot participate in the capping and stabilization of the AuNP. The pH ofthe reaction was varied from 3 to 11 to explore the effect of pH onparticle formation. The pH reactions were conducted at 10.0 g/L dextrinand 2 mM HAuCl₄, and incubated for 24 hours at 50° C. Below pH 7, noparticle formation was observed after 24 hours, and at pH 11 thereaction proceeded nearly instantly at room temperature. A negativecontrol with [AuCl₄]⁻ at pH 9.0 and without dextrin did not yieldparticle formation after 24 hours nor did a solution only adjusted withsodium hydroxide. The use of sodium hydroxide to adjust the pH insteadof sodium carbonate produced a purple-black precipitate with somemetallic gold film forming on the glassware. AuNPs with average particlediameters from 7.0±1.2 nm to 16.8±2.3 nm were generated within thebiological range of pH 7-10 in 24 hours. FIG. 3 shows the average sizesobtained with different pHs sodium carbonate adjusted and dextrinconcentration during generation.

The generation of AuNPs at pHs in the range of 7.0-11.0 with generationtimes from 1 minute to 6 hours, allows other biological materials, suchas DNA, to potentially be used as capping agents, with the possibilityof shortening the time or number of steps currently required for DNAfunctionalization. The faster reaction times and smaller particle sizesobserved could be explained by a similar mechanism to the four stepsynthesis proposed by Polte et al. (2010). The increased carbonateconcentration may cause faster and more complete nucleation of the[AuCl₄]⁻, [AuCl₃OH]⁻, [AuCl₂(OH)₂]⁻, [AuCl(OH)₃]⁻ or [Au(OH)₄]⁻ species.This faster rate reduces the available gold that would be used in steps3 and 4 for growth. By increasing the initial concentration of thereduction agents, faster nucleation may explain the observed increase ofthe reaction rate and observed decrease in the particle sizes. Reactionsless than pH 7 did not occur with sodium carbonate as the reductionagent. Carbonate has a pKa 6.33 and 10.35. At pH 9 the predominant formis bicarbonate, at pH 11 slightly more than 65% is carbonate, and at pH7 virtually no carbonate exists. Carbonate is a more reactive reducingspecies than bicarbonate and slow carbonate-bicarbonate equilibriumcould account for the longer generation times encountered. This couldexplain the rapid reaction rate at pH 11, where the carbonate is thepredominant specie in equilibrium and why at pH lower than 7 no reactionoccurs as nearly no carbonate exists in equilibrium.

Effect of Temperature:

The effect of temperature on the AuNP synthesis was explored todetermine if the reaction rate or particle size was influenced by thisfactor. Reactions were carried out at 50° C. and room temperature (21°C.) during 24 hours for concentrations of 20.0 g/L, 10.0 g/L and 2.5 g/Lof dextrin. At 50° C. particles were generated in all dextrinconcentrations after 6 hours. At room temperature, particle formationusing 10.0 g/L of dextrin was complete after 48 hours; using 2.5 g/L ofdextrin particle formation was approximately 70% complete after 48hours. Reaction completion was based on absorbance data (not shown).FIG. 4 shows the average particle diameter plotted against initialdextrin concentration at varying temperature. The particle size isaffected by both temperature and dextrin concentration. The averageparticle diameter increased with increasing temperature. Particleformation was observed at 100° C. in three minutes and completed after 7additional minutes on the bench top. The particles generated at 100° C.were not measured for this study as the reaction temperature wasnon-favorable for the stability of other possible biological cappingagents.

The initial reactions are clear after sodium carbonate adjustment,suggesting that nucleation has occurred and removed the Au³⁺ in freesolution (Kimling et al. 2006). Increases in generation temperature mayincrease the rate of disassociation of the capping molecule, dextrin inthis example. With higher temperatures, the partially uncapped AuNP ismore likely to interact with free gold from solution, step 3 assuggested by Polte et al. (2010), and result in a faster growth. A lowertemperature reduces the speed of growth in step 3 and the reaction maynot be fully completed, explaining the incomplete reaction at roomtemperature. This control of size and growth rate through temperaturevariation is expected to be a promising method for functionalizing theAuNPs during synthesis.

Stability:

The dextrin coated gold nanoparticles were stable for more than sixmonths at room temperature (21° C.) without protection from light. TheAuNPs were sensitive to low pH conditions, and when the system wastitrated to pH 3.5-4.0, a quick change in color was observed to darkpurple suggesting aggregation. Complete precipitation of the particlesoccurred after 12 hours at room temperature. After the color change, adeep purple particulate formed and precipitated. This insolubleprecipitate could not be resuspended by pH adjustment or sonication.Dextrin is an oligosaccharide of D-glucose, and D-glucose has a pKa˜12.3. The change in pH reverses the charge of the dextrin cappingmaterial thus eliminating the electrostatic interaction between dextrinand the AuNP core. When the particles were pelleted and dried, theresulting pellet could not be resuspended suggesting electrostaticinteractions in the aqueous medium are required for stability.

Functionalization:

The capping ligand exchange on citrate generated AuNPs for thiolated-DNAoligonucleotides is one of the more common attachment techniques (Hilland Mirkin 2006). The dextrin coated AuNPs were functionalized asdescribed with thiolated DNA in Hill and Mirkin (2006) and comparedagainst standard citrate reduced AuNPs. Dextrin AuNPs generated at 2.5g/L dextrin (size: 12.4 nm) and 10.0 g/L dextrin (size: 10.4 nm) wereevaluated for ligand exchange capabilities. Both sizes of dextrin coatedparticles were successfully functionalized with thiol-DNA-6-FAMoligonucleotides. The functionalization results are shown in FIG. 5.

Direct fluorescence measurement of the 6-FAM is limited because the AuNPcore quenches the signal. To measure the attached fluorophore, theDNA-6-FAM was ligand exchanged a second time with DTT to release thethiol-DNA-6-FAM. The recovered fluorescence after the second ligandexchange with DTT comes from the DNA attached to the AuNP cores anddemonstrates successful functionalization. The citrate AuNPs weretreated with the same ligand exchange procedure. The functionalizationefficiency of both sizes of dextrin AuNPs was comparable to the citrateAuNPs. After ligand exchange and release, the citrate AuNPs show greatercapping efficiency only when compared to the 10.4 nm dextrin AuNPs (77%of the citrate signal was recovered). The lower fluorescence recoveredwith the smaller particles was expected due to less available surfacearea for attachment (approximate 69% of the citrate particle surfacearea). The functionalization procedure is based on molar concentration,and smaller particles have less surface area for DNA attachment. For the12.4 nm dextrin AuNPs, the mean capping efficiency is equal to thecitrate AuNPs, where the dextrin particles have approximately 90% of theavailable surface area compared to the 13.0 nm citrate particles. Withcomparable DNA capping efficiencies, the dextrin AuNPs can be used as analternative to citrate AuNPs for DNA applications. Another potentialapplication of dextrin AuNPs is the exploration of simultaneousgeneration and functionalization, greatly reducing the functionalizationtime of current methodologies.

Proposed Mechanism of Generation:

The AuNP generation process observed with the dextrin and sodiumcarbonate system shows similarities with the citrate four step AuNPmechanism described by Polte et al. (2010). The initial nucleation(step 1) may be occurring during the pH adjustment when the solutionturns from yellow to clear, with sodium carbonate being the reductionagent. As the reaction is allowed to continue, a purple-black tint formssimilar to the aggregation and slow growth steps (steps 2-3) of themechanism proposed by Polte et al. (2010). The growth phase (step 4) isobserved during the 30-60 minute window where the solution initiallyturns from the purple tint to a red tint and then to the deep wine red,characteristic of the AuNP reaction.

Data from FTIR suggest that the capping is accomplished by non-oxidizeddextrin. There are no characteristic carboxylic acid absorbance peaks at1714 cm⁻¹, 1414 cm⁻¹, or 1294 cm⁻¹ (FIG. 6) suggesting that the cappingagent is not a dextrin with an aldose, ketose or a carbonate molecule.The generated AuNPs were adjusted to pH 4 to remove the charge on thedextrin. The pH 4 AuNP solution was agitated with chloroform to removethe unprotected gold. The dextrin remained in the water phase and theAuNP formed a solid black precipitate at the phase boundary betweenwater and chloroform. The dextrin-containing water phase was used forFTIR analysis. The stock dextrin (FIG. 6a ), autoclaved dextrin (FIG. 6b), and recovered dextrin (FIG. 6c ) all have similar FTIR peaks. Stockdextrin is a mixture of various chain lengths of repeated glucosesub-units which may allow the efficient AuNPs capping. The AuNP sizedistribution may be a result of the range in dextrin chain lengths (seehistograms in FIGS. 2a-2c ).

Summary:

The dextrin technique generated particles with a diameter between 5.9and 16.8 nm±1.6 nm based on dextrin concentration, pH, and temperature.Optimal AuNP synthesis was at 50° C., pH 9.0, 10.0 g/L of dextrin and 2mM of HAuCl₄ and resulted in particles of 10.6 nm±1.6 nm. The particlesremained soluble in water and stable for more than 4 months at roomtemperature. The dextrin capping agent was removed and the particleswere functionalized with thiolated ligands (DNA probes).

Dextrin coated particles may be used for many biological applicationsdue to the alkaline pH generation conditions. Alkaline generation ofdextrin AuNPs provides size control and a sugar capping agent that canbe removed for thiolated ligand exchange. The use of a polysaccharidefor capping provides a biocompatible coating for potential in vivo andin vitro use. Finally, the alkaline conditions potentially allowsimultaneous synthesis and functionalization procedures.

Example 2 Synthesis of Functionalized Gold Nanoparticles forTuberculosis Detection

This example describes the development of a DNA based biosensor todetect M. tuberculosis using thermophilic helicase-dependent isothermalamplification (tHDA) and dextrin coated gold nanopartcles (AuNPs) as anelectrochemical reporter. Dextrin coated AuNPs as in Example 1 are usedto create electrochemical labels and tHDA is used as an alternative toPCR for the DNA target amplification. The biosensor system is composedof gold nanoparticles (AuNPs) and amine-terminated magnetic particles(MPs) each functionalized with a different DNA probe that specificallyhybridizes with opposite ends of a fragment within the IS6110 gene,which is specific to Mycobacterium tuberculosis complex (MTC). Afterhybridization, the formed complex (MP-target-AuNP) is magneticallyseparated from the solution and the AuNPs are electrochemically detectedon a screen printed carbon electrode (SPCE) chip. The obtained detectionlimit is 0.01 ng/μl of isothermally amplified target (190 bp). Thisbiosensor system can be potentially implemented in peripherallaboratories with the use of a portable, handheld potentiostat.

Tuberculosis (TB) is the world's second deadliest infectious disease(1.7 million people die annually) and it has been identified as aleading cause of death among HIV-positive patients (WHO, 2009). Thestandard test for TB diagnosis is smear sputum microscopy, which isunable to identify half of the positive TB infections. Smear-negative TBdisease is highly common in HIV co-infected patients (Perkins et al.,2006). Among the novel detection methodologies are the nucleic acidamplification tests (NAATs). These have been extensively explored forthe rapid detection of M. tuberculosis. NAATs have high specificity andsensitivity, and can provide same day results (detection time: from 2-8h on processed specimens). However, NAATs require highly specializedpersonnel, expensive equipment, and are used only in proficientlaboratories that can afford reference reagents to monitor the assayperformance. Therefore, they are suitable for reference and peripherallaboratory implementation, but difficult to use in resource-constrainedsettings (Palomino, 2005).

Isothermal amplification techniques have been recently developed as analternative to polymerase chain reaction (PCR) for target DNAamplification and detection without the use of a thermocycler (Gill andGhaemi, 2008). Thermophilic helicase-dependent isothermal amplification(tHDA) utilizes a thermostable UvrD helicase to unwind the doublestranded DNA (dsDNA) and generate single stranded templates that areused for further polymerase amplification (Lixin et al., 2005). ThedsDNA separation and amplification are performed at the same temperature(60-65° C.) which makes this technique suitable for development ofpoint-of-care microbial detection systems, since a thermocycler is notrequired for DNA denaturation (95° C.) and amplification (Jeong et al.,2009). tHDA protocols have been developed for the detection of severalpathogens including: Helicobacter pylori (Gill et al., 2008),Clostridium difficile (Chow et al., 2008), S. aureus (Goldmeyer et al.,2008), N. gonorrhoeae (Lixin et al., 2005). tHDA has also been used inmicrofluidic chips with integrated sample preparation and amplificationto detect E. coli (Mahalanabis et al., 2010) and for the multiplexdetection of S. aureus and N. gonorrhoeae using a microarray on-chipamplification approach (Andresen et al., 2009).

Recently, nanomaterials have been introduced to enhance moleculardetection performance. Nanoparticles have been widely used over the lastdecade in the development of new diagnostic devices, especially quantumdots (QDs) and gold nanoparticles (AuNPs), (Azzazy et al., 2006).Sensitivity enhancement has been achieved with the use of nanoparticlesas tags or electrochemical labels. One of the most explored applicationsof AuNPs in clinical diagnostics is the use of AuNP probes (AuNP-ssDNAprobe) for the detection of DNA targets. The unique tunablephysicochemical properties of AuNPs, plus their good biologicalcompatibility, conducting capability, and high surface-to-volume ratiomake them ideal candidates for electronic signal transduction ofbiological recognition events in DNA-based electrochemical biosensingplatforms (Guo and Wang, 2007).

Nanoparticle Synthesis and Characterization:

The gold nanoparticles were synthesized following an alkaline basedmethodology as in Example 1 (see also Anderson et al., 2010,incorporated by reference in its entirety). A 50 mL solution of 2.5 g/Ldextrin and 2 mM gold chloride (HAuCl₄) was adjusted to pH 9 using 10%sodium carbonate and incubated at 50° C. with agitation in the dark for8 hours. After the reaction was completed, the solution changed frompallid yellow to dark red. The formed AuNPs (average size: 12.5 nm) werecharacterized using UV-Vis spectrophotometry (UV-VIS-NIR; Shimadzu) andtransmission electron microscopy (TEM) (JEOL 100CX). The amine-coatedmagnetic particles (average size: 1 μm) used in the biosensor systemwere commercially available (Sigma Aldrich Cat No. 17643-5 mL).

tHDA Primers and Hybridization Probes:

The tHDA primers were designed to specifically amplify a fragment of theIS6110 gene which is tuberculosis (TB) complex-specific (forward primer:5′ GAG CGT AGG CGT CGG TGA CAA AGG 3′ (SEQ ID NO: 2); reverse primer: 5′GCT TCG GAC CAC CAG CAC CTA ACC 3′ (SEQ ID NO: 3); GenBank: AJ242908.1(Mycobacterium tuberculosis IS6110 genes and partial plcD and Rv1758genes)). Specific fragments within the sequence (130-500 bp) have beenwidely used for PCR protocols targeting Mycobacterium tuberculosiscomplex (MTC) species (Dalovisio et al., 1996; Fernandez et al., 2009).The tHDA primers were designed considering the optimal conditions forisothermal amplification (IsoAmp II Universal tHDA kit, biohelix). Theobtained tHDA product (amplicon) was used as a template for thehybridization assay. Two different DNA probes (capture probe for MPs:5′-ss-AAA AAA AAA AAA GAG CGT AGG CGT CGG TGA-3′ (SEQ ID NO: 4) andreporter probe for AuNPs: 5′-GTG CTG GTG GTC CGA AGC AAA AAA AAAAAA-ss-3′ (SEQ ID NO: 5)) were also designed to specifically hybridizewith the fragment generated by the tHDA reaction. All oligonucleotideswere evaluated for specificity using the Primer-BLAST program (availablefrom the National Center for Biotechnology Information (NCBI) website).Primers and probes were designed using the Primer3 program (Rozen et al.2000) and purchased from IDT (Integrated DNA Technologies; CoralvilleIowa).

Nanoparticle Functionalization:

The dextrin coated AuNPs (˜12.5 nm in diameter) synthesized using thealkaline procedure were functionalized with a thiolated probe (TB 3′thiol; SEQ ID NO: 5) following a common methodology applicable forcitrate coated AuNPs ligand exchange (Anderson et al., 2010; Hill andMirkin, 2006). Briefly, the thiolated DNA probe (5 nmoles) was reducedwith DTT and purified using a SEPHADEX column (Nap-5, GE Healthcare).One milliliter of the dextrin coated AuNPs (AU=2 at A 520 nm) was mixedwith the purified probe solution. The dextrin molecules coating thesurface of the nanoparticles were exchanged for the thiolated DNA probeover a series of salting steps. After ligand exchange the particles werestored under refrigeration until use.

The amine coated MPs (˜1 μm in diameter) were functionalized with asecondary thiolated DNA probe (TB 5′ thiol; SEQ ID NO: 4) usingsulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate(sulfo-SMCC) as cross linker (Zhang et al., 2009). Briefly, the aminecoated MNPs (10 mg) were conjugated with sulfo-SMCC and incubated withthe DTT reduced and purified thiolated DNA probe (10 nmoles). Afterfunctionalization, sulfo-NHS acetate was used to block the unreactedlinker groups on the MP surface. After passivation, the particles werewashed and stored under refrigeration until use.

Helicase Dependent Isothermal Amplification (tHDA):

A synthetic target ssDNA containing the same oligonucleotide sequence ofthe IS6110 fragment (190 bp) was used (IDT Technologies). In order toevaluate the primers designed for the isothermal amplification, acommercially available kit for isothermal amplification was used (IsoAmpII Universal tHDA kit, biohelix). The amplification was conductedfollowing the tHDA conditions for one-step 50 μl tHDA reaction (1×annealing buffer II, 4 mM MgSO₄, 40 mM NaCl, 3.5 μl dNPT solution, 1 ngsynthetic target, 75 nM of each primer, 3.5 μl enzyme mix). The reactionwas covered with mineral oil (50 μl) to avoid evaporation. Theisothermal amplification reaction was also optimized using individualreactants: 1× thermo pol II buffer (provided with the polymerase), 4 mMMgSO₄, 3 mM dATP, 200 uM dNPTs, 20 U Bst DNA polymerase large fragment,100 ng thermostable helicase, 1 ng synthetic target DNA, 75 nM forwardprimer (105-F; SEQ ID NO: 2), 75 nM reverse primer (105-F; SEQ ID NO:3). Both enzymes were purchased from New England Biolabs. The totalreaction volume was 50 μl and same volume of mineral oil was added toavoid evaporation (Lixin et al., 2005; Vincent et al., 2004). Bothreactions were incubated in a regular heating block for 90 min at 65° C.(Eppendor Thermomixer R). After the reaction, the product was purifiedusing a silica membrane column (Miniellute, Qiagen) to eliminate theremaining primers. The purified product was serially diluted for thehybridization assay.

Hybridization Assay and Electrochemical Detection:

With reference to FIG. 7, the product obtained from the tHDA (target DNA10; 10 ng/μL-0.01 ng/μL) was denaturated at 95° C. for 10 min andallowed to hybridize with the capture DNA probe (TB 3′ thiol; SEQ ID NO:4) on the MPs (0.8 mg) (MNP with 1^(st) DNA probe 12) for 45 min at44.5° C. with continuous rotation to form a MP-target DNA complex 14.After magnetic separation and several washing steps to eliminate theunreacted target, AuNPs (40 μL) labeled with the reporter probe (TB 5′thiol; SEQ ID NO: 5) (AuNP with 2^(st) DNA probe 16) were added andincubated for 2 h at 44.5° C. with continuous rotation to form ahybridized sandwich complex 20 consisting of MNP-target DNA-AuNP (Zhanget al., 2009). The sandwich complex 20 was pulled and separated with amagnet 30. After several washing steps to eliminate the non-hybridizedAuNPs, the complex 20 was resuspended in 50 μL of water and transferredto a SPCE electrode 40 (Gwent electronic materials, Ltd.) forelectrochemical detection (FIG. 7). The SPCE 40 is composed of working(carbon) and reference (silver/silver chloride) electrodes. The solutionwas allowed to dry onto the carbon electrode for 30 min, and then 50 μLof 1M HCl solution was added to dissolve the AuNPs and generate Au³⁺ions 42. A constant 1.25 V was applied to the electrode for 2 min tooxidize the gold ions (potentiostat/galvanostat 263A and PowerSuitesoftware, Princeton Applied Research). Differential pulse voltammetry(DPV) was performed from 1.25V to 0.0V (with a step potential of 10 mV,modulation amplitude of 50 mV, and scan rate of 33.5 mV/s) to generatethe voltammogram as readout 44 produced by the reduced gold ions on theSPCE (Pumera et al., 2005).

Results—Nanoparticle Synthesis, Characterization and Functionalization:

The dextrin coated AuNPs were characterized using UV-Vis spectra andTEM. Absorbance peaks were obtained at 520 nm which is characteristic ofthe AuNPs production. The average particle diameter (12.4 nm) wasconfirmed by the TEM images. In this example, the dextrin-coated AuNPsare incorporated into a biosensor platform.

Helicase Dependent Isothermal Amplification (tHDA):

Successful amplification was obtained using the designed tHDA primers,synthetic target DNA and the tHDA kit following the conditions forone-step reaction (90 min of incubation at 65° C.) (IsoAmp II UniversaltHDA kit from biohelix). After the primer evaluation with thecommercially available kit, the isothermal amplification reaction wasalso optimized using individual reactants. Table 1 below shows theparameters used for the tHDA protocol design for the isothermal specificamplification of a IS6110 fragment and the corresponding FIG. 8 showsthe successful amplification products (bands) using individual reactantsfrom agarose gel electrophoresis.

TABLE 1 tHDA Parameters for Isothermal Amplification tHDA Parameter TBtHDA (IS6110 fragment) Optimal Amplicon size 105 pb 80-120 bp AmpliconTm 74° C. 68-75° C. Amplicon GC % 63%* 40% Primer size 24 b 24-33 basesPrimer Tm 71° C. 60-74° C. Primer GC % 62%* 35-60% *The specific regionselected within the IS6110 has high CG content.

Hybridization Assay and Electrochemical Detection of AuNPs:

Direct oxidation of AuNPs onto the carbon electrode surface wasoptimized at 1.25 V for 2 min obtaining a reduction peak of gold ionsbetween 0.30 and 0.35 V. FIG. 9a shows the DPV response obtained withdifferent DNA concentrations after hybridization, sandwich complexformation (MP-target-AuNP), magnetic separation and AuNPs dissolution.After the gold was dissolved and oxidized by the acidic solution, thegold ions were reduced between 0.30 and 0.35 V on the SPCE (FIG. 9a ).Three separate trials were run using four different DNA concentrations(0.01-10 ng/μl) plus a blank with no target DNA. Each sample was run intriplicates. A linear log-correlation was observed between the targetDNA concentration and the gold reduction peak (FIG. 9b ). The presenceor absence of the target was identified with a detection limit of 0.01ng/μl. Variability in the peak height was observed in different trialsbetween the two lowest concentrations detected (0.1 and 0.01 ng/μl).This variability may be the result of inter particle distances. At lowconcentrations, the particles are more dispersed and have moreaccessible surface area to interact with the acidic solution producingslightly higher reduction peaks. Therefore, the semi-quantitativedetection limit obtained using synthetic targets was 0.1 ng/μl.

These results represent the application of dextrin AuNPs aselectrochemical labels and the use of tHDA amplification products as DNAtargets. In order to evaluate the biosensor functionality, citrate AuNPsusing the capture probe according to SEQ ID NO: 5 and MPs using thecapture probe according to SEQ ID NO: 4 were used during initial runswith PCR products of the same synthetic target ssDNA corresponding tothe IS6110 fragment (190 bp) as targets for the hybridization reaction(data not shown). No difference was observed when using dextrin AuNPs inplace of citrate AuNPs as electrochemical labels. This was thehypothesized result since the DTT ligand exchange technique is intendedto remove all of the capping agent (i.e., dextrin or citrate,accordingly) on the surface of the AuNP in the oligonucleotidefunctionalization procedure. The particle's coating molecules areexchanged for thiolated DNA probes and the coating material is liberatedto the suspension liquid. After functionalization, the particles arecentrifuged and washed to eliminate the supernatant with unreactedmaterials. Since the elemental gold that is reduced in theelectrochemical detection is the same for citrate and dextrin coatedAuNPs, there is no effect of the coating material in the detectionsystem.

Summary:

This example describes the development and optimization of ananoparticle DNA-based biosensor to detect a tuberculosis specific DNAfragment using the electrochemical detection of gold nanoparticles. Inorder to make the platform more suitable for resource-constrainedsettings diagnostics, an isothermal amplification reaction was optimizedin lieu of PCR which requires a thermocycler. The isothermal helicasedependent amplification was successfully optimized using individualreactants and a regular heating block, which can be replaced with awater bath. The detection time with the proposed platform, includingamplification and hybridization is 6 hours; without considering samplepreparation (DNA extraction). The main advantages of the proposedplatform are that the detection system is not expensive, it can beportable and there is no need of a thermocycler due to the isothermalamplification.

The platform uses alkaline synthesized dextrin coated AuNPs aselectrochemical labels. No difference was observed when using dextrin orcitrate AuNPs as electrochemical labels in the detection system;nevertheless, the main advantage of using dextrin coated AuNPs is thesynthesis. The particles are produced under alkaline conditions (pH 9)and can be synthesized at room temperature. This opens the possibilityfor bio-molecule (e.g., DNA probes) functionalization during synthesis.The ligand exchange method traditionally used to functionalize citrateAuNPs with DNA probes requires 72 h post-synthesis (Hill and Mirkin2006).

Example 3 Synthesis of Acid-Functionalized Gold Nanoparticles

This example describes the synthesis of acid-functionalized AuNPs from adextrin-coated AuNP precursor. The acid-functionalized AuNPs arethemselves stable in solution provide a suitable platform for subsequentcovalent attachment of a biological probe moiety, for example a DNAprobe or antibody.

The method for forming the dextrin-coated AuNPs is similar to that ofExample 1. Briefly, a 50 ml aqueous solution of 20.0 g/L dextrin cappingagent and 10 g/L galactose supplemental saccharide is added to a 250 mlflack. Chloroauric acid (HAuCl₄.3H₂O) is added to the reaction mixtureas a gold source (final HAuCl₄ concentration: 2 mM), and the solution isadjusted to pH 9 using filter-sterile 10% sodium carbonate (Na₂CO₃). Thereaction flask is incubated in the dark (e.g., covered with aluminumfoil) at 50° C. with continuous shaking for 8-12 hours (e.g., overnight)with continuous shaking (e.g., 100 rpm). The result of this process is astabilized suspension of dextrin-coated AuNPs.

Acid-functionalization is performed as follows. 10 ml of 0.025 M sodiumdodecylsulfate (SDS) anionic surfactant is added to the suspension, andthe mixture is shaken (e.g., 200 rpm; not stirred, in an embodiment) atroom temperature for about 30 minutes. 10 ml of 0.087 mM11-mercaptoundecanoic acid (11-MUDA; final concentration: 1.24 μM) isthen added to the suspension, and the mixture is shaken (e.g., 200 rpm)at room temperature for about 1 hour. The result of this second processis a stabilized suspension of acid-functionalized AuNPs, where the thiolgroups of the 11-MUDA are stably adsorbed/bound to the AuNP goldsurface, and the outwardly pointing carboxylic acid groups of the11-MUDA provide hydrophilic groups to stabilize the aqueous suspensionand provide chemically reactive groups for the covalent attachment ofbiomolecules through various methods known in the art (e.g.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl-mediated reactionbetween the carboxylic acid groups and amino groups in a biomoleculeprobe; as in DeLong et al. 2010). In the context of thefunctionalization methods generally described above, the 11-MUDArepresents a binding pair member having a thiol immobilization moiety, acarboxylic acid binding moiety, the two of which are linked with anintervening hydrocarbon (alkylene) chain. Without wishing to be bound byany particular theory, it is believe that a surfactant such as SDS actsas an intermediate capping agent in that it assists in removal of thecarbohydrate capping agent and protects/stabilizes the uncharged andunprotected nanoparticle surface until a binding pair member (orimmobilization moiety thereof) such as 11-MUDA is able to bind to thesurface and re-stabilize the suspension.

Example 4 Synthesis of Gold-Coated Magnetic Nanoparticles

This example provides a gold-coated iron oxide (Fe₃O₄) magnetic particleusing dextrin as a capping agent that could be used as an extraction anddetection tool for pathogenic cells in an electrochemical biosensorsystem. Other gold-coated Fe₃O₄ nanoparticles have been synthesizedusing citrate as a capping agent. The dextrin-capped gold magneticnanoparticles produced particles with a stronger spectrophotometricabsorption spectrum relative to citrate-capped particles. The gold peakwas confirmed using a potentiostat. For comparison, the gold-coatedFe₃O₄ nanoparticles were functionalized with oriented antibodies usingprotein A and non-oriented antibodies through nonspecific adsorption.These were used to magnetically separate Escherichia coli O157:H7 from abroth matrix. The protein A and antibody concentrations were varied todetermine the most effective concentration for the conjugate to maximizeE. coli O157:H7 extraction. The nanoparticle-bacteria complex wasapplied to a modified screen printed carbon electrode. Voltage wasapplied and current was measured. A current peak was observed at about0.3 V to about 0.4 V, signifying the presence of bacteria and thusconfirming the extraction. The results indicate that dextrin-capped goldmagnetic nanoparticles could be used as an extraction and detection toolto report the presence of target bacteria in the biosensor.

The following materials/stock solutions (in distilled/sterile water)were prepared for synthesis of the gold-coated magnetic iron oxidenanoparticles: iron (II) chloride tetrahydrate (FeCl₂.4H₂O), iron (III)chloride hexahydrate (FeCl₃.6H₂O), hydrochloric acid (HCl; 0.5 M),sodium hydroxide (NaOH; 1.5 M), tetramethylammonium hydroxide (TMAOH;0.1 M), hydroxylamine hydrochloride (NH₂OH.HCl; 0.2 M), dextrin (25g/L), chloroauric acid (HAuCl₄; 0.4 g/50 mL), and sodium carbonate(Na₂CO₃; 10% w/v).

The magnetic iron oxide (Fe₃O₄) nanoparticles were synthesized with thefollowing procedure. [1] In a 50 mL beaker, dissolve 5.4 g of FeCl₃.6H₂Oand 2.0 g of FeCl₂.4H₂O in 25 mL of 0.4M HCl. [2] In a 400 mL beaker,add the iron chloride solution dropwise to 250 mL of 1.5M NaOH to formFe₃O₄ nanoparticles as a black precipitate. [3] Collect the Fe₃O₄precipitate using a magnet. [4] Dispose of supernatant and wash theprecipitate twice with sterile water. [5] Wash the precipitate twicewith 0.1M TMAOH. [6] Add 250 mL of 0.1M TMAOH to the Fe₃O₄ precipitate(for storage as a solution/suspension).

The magnetic iron oxide (Fe₃O₄) nanoparticles were then coated with adextrin-capped gold layer using the following procedure. [1] In asterile 50 mL tube, combine 17.7 mL sterile H₂O, 20 mL of 25 g/Ldextrin, and 5 mL of 0.4 g/50 mL HAuCl₄. [2] Adjust the solution to pHof 9 using Na₂CO₃. [3] Add 3.33 mL of the Fe₃O₄ nanoparticle suspensionto the dextrin/gold solution. [4] Add 4.8 mL of 0.2M NH₂OH.HCl as anadditional reducing agent. [5] Incubate in rotisserie in the dark at 50°C. with continuous rotation for 6 hours. [6] Separate thedextrin-capped, gold-coated Fe₃O₄ nanoparticles from thereaction/reduction medium using a magnet (e.g., to separate the magneticnanoparticle product from non-magnetic gold nanoparticles formed duringthe reduction process but without a Fe₃O₄ nanoparticle core/nucleationsite). [7] Discard supernatant and wash twice with sterile water. [8]Repeat steps 1-4. [9] Incubate in rotisserie in the dark at 25° C. withcontinuous rotation overnight. [10] Separate the dextrin-capped,gold-coated Fe₃O₄ nanoparticles from the reaction/reduction medium usinga magnet. [11] Discard supernatant and wash twice with sterile water.[12] Store the dextrin-capped, gold-coated Fe₃O₄ nanoparticles in 50 mLof sterile water in capped tube. FIG. 10 is a TEM image of exampledextrin-capped, gold-coated magnetic nanoparticles formed using thisprocedure (e.g., showing small particles down to about 5 nm to 8 nm,large particles up to about 30 nm to 50 nm, and an average/medianparticle size of about 10 nm to 15 nm).

The dextrin-capped, gold-coated Fe₃O₄ nanoparticles were thenfunctionalized with anti-E. coli O157:H7 antibodies either (i) by directadsorption/ligand exchange of the antibodies onto the gold surface ofthe magnetic nanoparticles or (i) by direct adsorption/ligand exchangeof protein A/G onto the gold surface of the magnetic nanoparticlesfollowed by incubation with the antibodies to immobilize and properlyorient the antibodies on the magnetic nanoparticles. Magnetic separationwas used to remove unbound antibodies and the functionalized gold-coatedmagnetic nanoparticles were re-suspended in phosphate buffered saline(PBS).

FIG. 11 illustrates a process for capturing, isolating, andconcentrating E. coli O157:H7 (target/analyte 10) from a broth matrix(sample 11) using the functionalized gold-coated magnetic nanoparticles16. In particular, E. coli O157:H7 extraction was performed by mixing 30μl of the functionalized gold-coated magnetic nanoparticles antibodyconjugate 16, 30 μl of a bacterial dilution 10/11, and 240 μl of PBS.Solutions were placed in a rotator for 30 minutes in order for theantibodies to bind with the bacteria, forming a complex/conjugate 20.Bacteria that successfully attached to the functionalized nanoparticlesas the complex 20 were magnetically separated from unbound bacteriausing a magnet 30 (FIG. 11). Conjugates 20 of bound bacteria 10 and thefunctionalized nanoparticles 16 were washed twice with PBS andre-suspended in PBS. The capture efficiencies of the functionalizednanoparticles 16 were determined by surface plating the bound cells onMacConkey agar and incubating at 3TC for 24 hours.

Results:

Electrochemical analysis of the dextrin-capped, gold-coated Fe₃O₄nanoparticles produced a higher and sharper electrochemical signal thancorresponding citrate-capped, gold-coated Fe₃O₄ nanoparticles (FIG. 12).As illustrated in FIG. 12, the dextrin-capped nanoparticles exhibit arelatively narrow electrochemical gold reduction peak at about 0.3 V to0.4 V, making the particles a suitable platform/electrochemical signaltransducer for detection and/or identification of a conjugated targetanalyte (i.e., the gold reduction peak corresponding to a target analytedetection is not subject to substantial signal interference or overlapfrom other system components). In contrast, the electrochemical responseof corresponding citrate-capped nanoparticles in FIG. 12 exhibits arelatively wide peak over the range from about 0.2 V to 0.9 V, with theadditional citrate contribution to the signal making it difficult toreliably and/or accurately detect a gold reduction peak corresponding tothe presence of the target analyte (e.g., potentially resulting in falsepositive, false negative, and/or inaccurate quantitative analytedeterminations). Fluorescence-tagged antibodies were used and detectedto verify functionalization of the magnetic nanoparticles (FIG. 13). Therecovery of bacteria from broth at concentrations ranging from about 10²CFU/mL to 10⁴ CFU/mL was found to have a high extraction/captureefficiency of about 75% to about 80% (FIG. 14).

Example 5 Synthesis of Functionalized Gold Nanoparticles for E. coliDetection

This example describes the formation and testing of ananoparticle-labeled biosensor designed for the rapid detection ofEscherichia coli O157:H7 in broth. Compared to conventional cultureplating methods, the biosensor reduced the detection time from 2-4 daysto less than one hour without complicated manipulation. Polymer-coatedmagnetic nanoparticles (MNPs) were conjugated with monoclonal antibodiesto separate target E. coli O157:H7 cells from broth samples.Carbohydrate-capped gold nanoparticles (AuNPs) were conjugated withpolyclonal antibodies and were then introduced to the MNP-target complexto form a sandwich MNP-target-AuNP (e.g., where the AuNP serves asanalyte label/electrochemical transducer). By measuring the amount ofgold nanoparticles through an electrochemical method, the presence andthe amount of the target bacteria were determined. The results showed asensitivity of 10¹ cfu/ml with a linear range of 10¹-10⁶ cfu/ml. Thenanoparticle-labeled biosensor can be used for the rapid detection ofinfectious agents for public health, biodefense, and food/water safety.

Reagents and Materials:

Two kinds of nanoparticles were synthesized: magnetic nanoparticles(MNPs) and gold nanoparticles (AuNPs). Aniline, iron (III) oxidenanopowder, ammonium persulfate, methanol, and diethyl ether were usedfor the synthesis of the MNPs. Gold (III) chloride trihydrate (Aldrich,Mo.) and dextrin (Fluka, Mo.) were used for the synthesis of goldnanoparticles under alkaline conditions as in Example 1 above. Sodiumsulfide, 3-mercaptoacetic acid, lead nitrate and were used for thesynthesis of PbS nanoparticles. MNPs were functionalized with monoclonalanti-E. coli O157:H7 antibodies obtained from Meridian Life Science,Inc. (Saco, Me.). AuNPs were conjugated with polyclonal anti-E. coliO157:H7 antibodies from Meridian Life Science, Inc (Saco, Me.). ProteinA from Staphylococcus aureus was used as the linkage agent for AuNP andantibody conjugation. TRITON-X100, phosphate buffered saline (PBS),casein, bovine serum albumin (BSA) and sodium phosphate (dibasic andmonobasic) were obtained from Sigma-Aldrich (St. Louis, Mo.). PBS buffer(0.01 M, pH 7.4), PBS buffer with 0.05% (w/v) TRITON-X100, phosphatebuffer (0.1 M sodium phosphate, pH 7.4), PBS buffer with 0.01% casein,PBS buffer with 0.1% (w/v) BSA were prepared with deionized water from aMillipore DIRECT-Q system.

Bacterial Culture:

E. coli O157:H7 Sakai strain was obtained from the Nano-biosensors Labcollection at Michigan State University. The colonies from frozen(stored at −70° C.) culture were grown on trypticase soy agar (BDBiosciences, MD) plates. A single colony was isolated and inoculated intryptic soy broth (BD Biosciences, MD) and grown overnight at 37° C. Onemilliliter of the liquid culture was transferred to another tube oftryptic soy broth and incubated overnight at 37° C. One milliliter ofthis liquid culture was transferred to a new tube of broth and incubatedat 37° C. for 6 h before each experiment. The serial dilutions ofbacterial culture were prepared using 0.1% (w/v) peptone water(Fluka-Biochemika, Switzerland) before each experiment. Viable cellswere enumerated by microbial plating on MacConkey agar with sorbitol (BDBiosciences, MD).

Apparatus:

Electrochemical measurement was performed with apotentiostat/galvanostat (263A, Princeton Applied Research, MA) with thesoftware operating system (PowerSuite, Princeton Applied Research, MA)on a computer connected to the potentiostat. The measurement wasperformed by introducing each sample onto a screen-printed carbonelectrode (SPCE) chip (Gwent Inc. England). As similarly described abovein Examples 2 and 4, the SPCE chip consists of a working electrode(carbon) and a counter and reference electrode (silver/silver chlorideelectrode). One hundred microliters of each sample were introduced tothe electrode area on the SPCE chip.

Nanoparticle Synthesis:

Polyaniline (PANI) coated magnetic nanoparticles were synthesizedaccording to Alocilja et al. U.S. Pat. No. 8,287,810 and U.S.Publication No. 2009/0123939 (incorporated herein by reference in theirentireties). Briefly, 50 ml of 1 M HCl, 10 ml of water and 0.4 ml ofaniline monomer were mixed in a flask, and then 0.65 g of iron (III)oxide nanopowder were added to the solution to maintain a final γ-Fe₂O₃:aniline weight ratio of 1:0.6. The mixture was put in a beaker filledwith ice and sonicated for 1 h. The solution was stirred while it wasstill on ice. During the stirring, ammonium persulfate (1 g of ammoniumpersulfate in 20 ml deionized water) was added to the solution slowlyfor 30 min. The solution was stirred for another 1.5 h. After thereaction, the solution was filtered using 2.5 μm filter paper and washedwith 20% methanol. Hydrochloric acid (1 M) was used to wash untilfiltrate became clear, followed by washing with 10 ml 20% methanol. Thefiltrate was filtered again using a 1.2 μm filter paper. Twenty percentmethanol solution was added to the filter. The HCl and methanol wash wasrepeated. The nanoparticles on the filter paper were left under a fumehood to dry for 24 h at room temperature and stored in vacuum desiccatorafter drying.

Dextrin-capped, gold nanoparticles were synthesized under alkalinecondition according to the method of Example 1 above. Briefly, 20 ml ofdextrin stock solution (25 g/l) and 20 ml of sterile water were mixed ina 50 ml sterile orange cap tube (disposable). Five milliliters of HAuCl₄stock solution (8 g/ml) were then added, and the pH of the solution wasadjusted to 9 with sterile 10% (w/v) Na₂CO₃ solution. The final volumewas brought to 50 ml with pH 9 water. The reaction was carried out byincubating the solution in a sterile flask in the dark at 50° C. withcontinuous shaking (100 rpm) for 6 h. A red solution was obtained at theend of the reaction. The final concentration of the aqueous suspensionof dextrin-capped AuNPs was 10 mg/ml.

Nanoparticle Functionalization:

The MNPs were functionalized with monoclonal antibody (mAb) to E. coliO157:H7. MNPs (2.5 mg) were suspended in 150 μl of 0.1M phosphatebuffer, and sonicated for 15 min. Monoclonal anti-E. coli O157:H7antibody (2.5 mg/ml, 100 μl) was added to the suspension, and hybridizedon tube rotor for 5 min. Twenty five microliters of PBS (0.1 M) wereadded. Then the conjugation was carried on for 55 min on the tube rotor.The MNPs were separated from the solution by magnetic separation, andblocked by adding 250 μl of 0.1M tris buffer with 0.01% casein for 5 minincubation. This step was repeated three times, and the suspension wasput on tube rotor for 60 min hybridization at the last time. Finally,the MNPs were magnetically separated and resuspended in 2.5 ml of 0.1MPhosphate buffer. The MNP-mAb conjugate was stored at 4° C. before use.In a different study (not shown), the MNP was validated in about 40related E. coli O157:H7 strains and 30 unrelated bacterial strains withan inclusivity of about 94%.

Gold nanoparticles were conjugated with polyclonal antibody (pAb) to E.coli O157:H7 through a protein A linkage. Two hundred microliters of 5mg/ml dextrin-capped AuNPs were put into a 2 ml microcentrifuge tube andsonicated for 10 minutes. Then the suspension was centrifuged for 6 minat 13,000 rpm. The supernatant was removed after the centrifugation. Tomodify the surface of the AuNPs, protein A (0.25 mg/ml) in PBS was usedto resuspend the AuNPs. The conjugation was conducted by rotating themixture for 60 min. The modified AuNPs were separated from thesuspension by centrifugation for 6 min at 13,000 rpm. The nanoparticleswere washed by adding 200 μl of 0.01 PBS and centrifuged. After removingthe supernatant, 100 μl of 1 mg/ml antibody and 100 μl PBS were added tothe tube and mixed for 60 min by rotating. After separating theAuNP-antibody (AuNP-pAb) conjugates, two hundred microliters of theblocking agent were added to the tube. The mixture was rotated for 30min. Finally, the AuNP-pAb conjugates were separated from the suspensionby centrifugation, and the final suspension of the conjugates in PBS wasstored at 4° C. Similar to Example 4 above, the dextrin capping agent,not having been entirely removed by an explicit ligand exchange process,partially remained as a capping/stabilizing agent for the AuNP-pAbconjugates (e.g., the adsorbed protein A and antibodies may displacesome (but not all) of the capping agent from the AuNP surface).

Bacteria Detection:

The process for the capture, separation, and detection of the targetpathogen 10 is illustrated in FIG. 15. The blank control for the testswas peptone water in the same volume as the sample 11. Firstly, 400 μlof PBS, 50 μl of cell dilution (or peptone water for the control) and 50μl of MNP-mAb conjugates 12 were combined in a 2 ml sterile tube. After15 min hybridization, PBS (55 μl, 0.01 M) with 0.1% BSA was added to themixture as a blocking agent. Then, the MNP-E. coli complexes 14 weremagnetically separated from the sample matrix 11 solution using a magnet30 and resuspended in 450 μl of PBS. Secondly, the dextrin-cappedAuNP-pAb conjugates 16 were introduced to the system, followed by 15 minhybridization to form MNP-mAb-target-pAb-AuNP complexes/conjugates 20.After washing the complexes 20 once with 0.01 M PBS, the complexes 20were resuspended in 500 μl of PBS with 0.05% TRITON-X100, and left standfor a few minutes. Finally, the complexes 20 were suspended in 500 μl ofPBS. One hundred microliters of the suspension were plated on SMAC forcell counting. The rest were magnetically separated from the supernatant(400 μl).

Electrochemical Measurement:

The target bacteria were detected by measuring the electrochemicalsignal of AuNPs. Each sample obtained as described above (complexes 20magnetically separated from supernatant) was combined with 100 μl 1 MHCl (to generate Au³⁺ ions for detection as the analyte/bacterial labelfrom the AuNPs) and was introduced to the SPCE chip 40. An oxidationpotential of 1.4 V vs. Ag/AgCl was applied to the working electrode.After oxidation, a differential pulse voltammetric (DPV) measurement wasperformed. The scan was from −1.5 V to 1.5 V. The potential and currentswere recorded. All measurements were performed at room temperature. Eachsample was measured three times. At least three samples for eachconcentration of bacteria were tested.

Results:

FIG. 16 shows typical DPV sensorgrams of native AuNPs and nativemagnetic nanoparticles (MNPs). The current peak for AuNPs is at 0.25Vand MNPs is at 0.58V. FIG. 17 shows typical DPV sensorgrams for thedetection of E. coli O157:H7 at different cell concentrations (10², 10⁴,and 10⁶ cfu/ml) relative to a negative control. The sensorgrams show awider curve which seems to include both AuNPs and MNPs. For theanalysis, the peak current to the left (representing AuNPs) was chosenfor signal reporting. As shown in the FIG. 17, the peak current forAuNPs increases with increasing cell concentration. FIG. 17 also showsthe formation of the MNP-cell-AuNP complex. The amount of target cellsdetected is proportional to the amount of AuNPs. The lowest measuredcell concentration in FIG. 17 was detectable at 10² cfu/ml. FIG. 18 is aplot of the peak (gold) current vs. cell concentration for theMNP-cell-AuNP complex, and it shows a linear relationship between thepeak DPV current and the log cell concentration between 10¹ and 10⁶cfu/ml. The results verify that AuNP could be used for labeling thetarget cells, and the magnetic separation is effective.

Example 6 Synthesis of Functionalized Gold Nanoparticles with aNanotracer for E. coli Detection

This example describes the formation and testing of a nanotracer-labelednanoparticle designed for the rapid detection of Escherichia coliO157:H7. The detection system includes antibody-functionalized,polymer-coated magnetic nanoparticles (MNP-mAb) as described in Example5 for the capture and separation of target E. coli O157:H7 cells from asample (broth) matrix. Dextrin-capped, antibody-functionalized goldnanoparticles (AuNP-pAb, with the polyclonal antibody pAb for E. coliO157:H7) as described in Example 5 are further functionalized to includemetallic nanotracer (NT) particles (lead sulfide, PbS) attached to theAuNP substrate via barcode DNA/oligonucleotide (bDNA) as generallydescribed in Alocilja et al. U.S. Publication No. 2011/0171749(incorporated herein by reference in its entirety). The resultingfunctionalized gold nanoparticle (NT-bDNA-AuNP-pAb) is then used as ananalyte label in which the lead (Pb²⁺) of the NT serves as a detectablemoiety for the electrochemical detection of the E. coli analyte (e.g.,by square wave voltammetry).

The dextrin-capped, antibody-functionalized gold nanoparticles(AuNP-pAb) as described in Example 5 are further functionalized byattaching a thiolated, amine-functional bDNA oligonucleotide(5′-[amino]-GTC AGT CAG TCA GTC AGT CA-[thiol]-3′ (SEQ ID NO: 6)). ThebDNA is attached to the AuNP surface at the 3′-end via a thiol linkageusing the DTT ligand exchange procedure generally described in Examples1 and 2 above (i.e., thus essentially removing the dextrin capping agentfrom the intermediate bDNA-AuNP-pAb composition). Lead sulfide (PbS) NTparticles are formed using the method of Salavati-Niasari et al. (2012).Briefly, lead nitrate (Pb(NO₃)₂) and thioglycolic acid (HSCH₂COOH) arereacted to form complexed PbS NT nanoparticles (e.g., containingcarboxylic groups for further functionalization). The PbS NTnanoparticles are then attached to the 5′-end of the bDNA using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) andN-hydroxysuccinimide (NHS) linkers as generally known in the art to formthe resulting analyte label (NT-bDNA-AuNP-pAb).

FIG. 19 illustrates a process for the capture, separation, and detectionof a target pathogen 10 similar to that of Example 5 and FIG. 15.Briefly, the MNP-mAb 12 nanoparticles are added to a sample volumecontaining the E. coli target analyte 10 to hybridize and form MNP-E.coli complexes 14. The MNP-E. coli complexes 14 are magneticallyseparated from the sample matrix solution using a magnet 30 and thenresuspended in solution for combination with the analyte label 16NT-bDNA-AuNP-pAb, followed by hybridization to form the triplexconjugate 20 MNP-E. coli-AuNP including the NT 18 as PbS nanoparticlestethered to the AuNP nanoparticle core via the bDNA. The triplexconjugates 20 are then magnetically separated/concentrated from thesuspension for nanotracer 18 dissolution, thus releasing metalnanotracer ions 42 (e.g., Pb²⁺) that can be detected and correlated tothe presence of the analyte 10 by any suitable electrochemical method,such as square-wave voltammetry (e.g., using an SPCE biosensor 40 asillustrated). Other details related to the use and detection of barcodeDNA nanotracer analyte labels may be found in Alocilja et al. U.S.Publication No. 2011/0171749.

Because other modifications and changes varied to fit particularoperating requirements and environments will be apparent to thoseskilled in the art, the disclosure is not considered limited to theexample chosen for purposes of illustration, and covers all changes andmodifications which do not constitute departures from the true spiritand scope of this disclosure.

Accordingly, the foregoing description is given for clearness ofunderstanding only, and no unnecessary limitations should be understoodtherefrom, as modifications within the scope of the disclosure may beapparent to those having ordinary skill in the art.

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

Throughout the specification, where the compositions, processes, kits,or apparatus are described as including components, steps, or materials,it is contemplated that the compositions, processes, or apparatus canalso comprise, consist essentially of, or consist of, any combination ofthe recited components or materials, unless described otherwise.Combinations of components are contemplated to include homogeneousand/or heterogeneous mixtures, as would be understood by a person ofordinary skill in the art in view of the foregoing disclosure.

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What is claimed is:
 1. A method for forming reduced metal nanoparticles,the method comprising: (a) providing an aqueous medium, the aqueousmedium comprising (i) water and (ii) metal ions in solution in thewater; and (b) reducing the metal ions in the aqueous medium at aneutral or alkaline pH value in the presence of at least one of a linearand a branched carbohydrate capping agent for a time sufficient to forma plurality of reduced metal nanoparticles as a suspension stabilized inthe aqueous medium with the carbohydrate capping agent adsorbed on outersurfaces of the reduced metal nanoparticles; wherein the carbohydratecapping agent is present in the aqueous medium prior to reaction at aconcentration ranging from 1 g/L to 100 g/L.
 2. The method of claim 1,wherein the plurality of reduced metal nanoparticles has an averageparticle size ranging from 5 nm to 50 nm.
 3. The method of claim 2,wherein the metal ions comprise gold ions and the plurality of reducedmetal nanoparticles comprise gold nanoparticles.
 4. The method of claim2, wherein the aqueous medium further comprises a counter ion insolution in the water from a dissolved metal ionic compound providingthe metal ions.
 5. The method of claim 2, wherein the pH value of theaqueous medium in (b) ranges from 7 to
 12. 6. The method of claim 2,comprising performing (b) at a temperature ranging from 20° C. to 100°C.
 7. The method of claim 2, wherein the aqueous medium in (b) furthercomprises a combined reducing agent for reducing the metal ions andpH-adjusting agent for maintaining the neutral or alkaline pH value ofthe aqueous medium.
 8. The method of claim 2, wherein the carbohydratecapping agent comprises an oligosaccharide having 3 to 100 saccharideresidues.
 9. The method of claim 2, wherein the carbohydrate cappingagent comprises a plurality of oligosaccharides having a distribution oflengths with a number-average of saccharide residues ranging from 10 to100.
 10. The method of claim 2, wherein the carbohydrate capping agentcomprises one or more glucose residues.
 11. The method of claim 2,wherein the carbohydrate capping agent is in a substantiallynon-oxidized form.
 12. The method of claim 2, wherein the carbohydratecapping agent comprises dextrin.
 13. The method of claim 2, comprisingperforming the metal ion reduction in (b) in the presence of at leastone of a monosaccharide and a disaccharide in addition to thecarbohydrate capping agent.
 14. The method of claim 2, wherein: (i) thecarbohydrate capping agent comprises at least one of a monosaccharideand a disaccharide; and (ii) (b) comprises performing the metal ionreduction in (b) in the presence of at least one non-carbohydratecapping agent in addition to the carbohydrate capping agent.
 15. Themethod of claim 2, wherein the carbohydrate capping agent has aconcentration in the aqueous medium in (b) selected to control one ormore size parameters of the plurality of metal nanoparticles.
 16. Themethod of claim 2, wherein the plurality of reduced metal nanoparticleshas an average particle size ranging from 8 nm to 50 nm.
 17. The methodof claim 16, wherein the plurality of reduced metal nanoparticles has anormal size distribution with a standard deviation of 25% or lessrelative to the average particle size of the distribution.
 18. Themethod of claim 2, wherein at least some of the carbohydrate cappingagent is present as a layer on an outer surface of each reduced metalnanoparticle.
 19. The method of claim 2, wherein: (i) the aqueous mediumin (b) further comprises a binding pair member comprising (A) animmobilization moiety for immobilizing the binding pair member onto areduced metal nanoparticle and (B) a binding moiety capable of bindingto a target analyte or a second binding pair member; and (ii) (b) isperformed for a time sufficient in the presence of the binding pairmember to additionally immobilize the binding pair member on an outersurface of a reduced metal nanoparticle via the immobilization moiety.20. The method of claim 19, wherein the immobilization moiety of thebinding pair member comprises a carbohydrate moiety conjugated to thebinding moiety.
 21. The method of claim 2, wherein the carbohydratecapping agent has at least 10 saccharide residues.
 22. The method ofclaim 2, wherein the reduced metal nanoparticles have a spherical shape,and the plurality of reduced metal nanoparticles has a number-averageparticle size ranging from 8 nm to 30 nm.
 23. A method for formingreduced gold nanoparticles, the method comprising: (a) providing anaqueous medium, the aqueous medium comprising (i) water and (ii) goldions in solution in the water; and (b) reducing the gold ions in theaqueous medium at a pH value ranging from 8 to 11 in the presence of atleast one of a linear and a branched dextrin capping agent for a timesufficient to form a plurality of reduced gold nanoparticles as asuspension stabilized in the aqueous medium with the dextrin cappingagent adsorbed on outer surfaces of the reduced gold nanoparticles;wherein the plurality of reduced gold nanoparticles has an averageparticle size ranging from 5 nm to 15 nm, the dextrin capping agent hasat least 10 saccharide residues, and the dextrin capping agent ispresent in the aqueous medium prior to reaction at a concentrationranging from 1 g/L to 100 g/L.
 24. The method of claim 23, furthercomprising performing the metal ion reduction in (b) in the presence ofgalactose in addition to the dextrin capping agent.
 25. The method ofclaim 23, wherein the dextrin capping agent comprises a plurality ofoligosaccharides having a distribution of lengths with a number-averageof saccharide residues ranging from 10 to
 100. 26. The method of claim23, wherein the reduced gold nanoparticles have a spherical shape, andthe plurality of reduced gold nanoparticles has a number-averageparticle size ranging from 8 nm to 15 nm.